HK1201567B - Rapid genotyping analysis for human papillomavirus and the device thereof - Google Patents

Description

Rapid genotype identification and analysis of human papilloma virus and device thereof

This application claims priority to U.S. patent application serial No. 12/770034 filed on day 29, 4/2010, partially succeeding U.S. patent application serial No. 11/398433 filed on day 4, 4/2006 and partially succeeding U.S. patent application serial No. 10/291168 filed on day 11, 7, 2002, while claiming benefit of U.S. patent application serial No. 60/345948 filed on day 11, 7, 2001. U.S. patent application serial No. 11/398433 is also U.S. patent application serial No. 10/293248 filed in part for 11/9 in 2002. The contents of the foregoing application are hereby incorporated by reference into this application in their entirety.

Technical Field

The present invention relates to the identification of multiple genotypes of human papilloma virus.

Background

Identification of Human Leukocyte Antigens (HLA)

Matching donors and recipients by accurate HLA typing in organ or bone marrow transplantation (4) is critical to preventing the development of acute Graft Versus Host Disease (GVHD). HLA typing is typically accomplished by standard serological typing (2). Recent studies have shown that DNA typing provides more accurate and positive results (7, 9, 8). HLA-DQ, HLA-DR and HLA-DP typing test results are used for high precision matching, which has been a necessary approach for selection of potential organ donors (3). It has been previously reported that HLA genotyping can be performed by polymerase chain reaction (SSP-PCR) amplification using sequence-specific primers. However, due to the high polymorphism of HLA-DQ, DR, and DP sites, the number of Sequence Specific Primers (SSPs) required will be as many as several hundred, beyond the limit of multiplex PCR amplification, and thus efficient PCR amplification cannot be performed. To ensure that the results of HLA genotyping are clinically practical, multiple assays must be set up, each with 50 to 100 independent PCR reactions. Kits are now available on the market, which comprise an amplification process, requiring 96 separate PCR reactions followed by analysis of the size of the fragments separated by gel electrophoresis. This is not only time and money consuming, but is also very error prone because the reaction set-up is complicated and there is also uncertainty in determining the size of the fragment in gel electrophoresis. Therefore, DNA sequencing is still considered the first choice for accurate typing of HLA genes. Unfortunately, pseudogenes can also be co-amplified in the Polymerase Chain Reaction (PCR) due to high homology in sequence, making high resolution HLA genotyping by DNA sequencing alone more difficult and expensive. U.S. patent nos. 5471547 and 6020187, issued to mr. JWO Tam, disclose a rapid annealing process that can be performed on cost-effective equipment for accurate mutation detection, genotyping, and fingerprinting. The invention discloses a method for rapidly analyzing HLA locus DP, DR and DQ beta sequences by using an ASO oligonucleotide probe in an improved diversion mode.

DNA fingerprinting for rapid identification of humans and organisms

In 1985, Restriction Fragment Length Polymorphism (RFLP) -based DNA fingerprinting was first applied for human identity identification (12) and subsequently began to be applied for identification of other organisms. In practical applications, DNA fingerprinting technology has been widely accepted as the best forensic tool for use in criminal cases to identify criminal suspects, disputes, establish or verify the identity of a person. The time-consuming RFLP method has been gradually replaced by high-throughput automated processes. Currently, single cell level identification has been achieved by analyzing the number of Short Tandem Repeats (STRs) in 10, 16, 18 or more loci of the human genome using PCR amplification (found in 1991 (11)). However, both STRs and Variable Numbers of Tandem Repeats (VNTRs) are relatively expensive because these methods require the use of complex equipment, as well as manually intensive and time consuming procedures such as Southern blot hybridization. Spontaneous mutation (10) may also reduce accuracy and certainty of the final identification result. Furthermore, STR data indicate that the frequency of mutations, particularly in cancer patients, is not uncommon. Therefore, new alternatives need to be found. Single Nucleotide Polymorphism (SNP) genotyping is a higher resolution method in forensic or personal identification based on the selection of a certain number of SNP sites at unlinked sites, where the mutation frequency of each site is lower than in VNTR or STR systems. The invention discloses a method for rapidly and definitely identifying individual biological characteristics including human beings, animals, plants or other organisms by utilizing SNP genotype identification.

HPV genotyping identification

Human Papilloma Virus (HPV) attacks mucosal cells with high diversity, with approximately 200 different genotypes being reported worldwide with varying prevalence among different populations. Human papillomaviruses are classified into high-risk strains (HR) and low-risk strains (LR) according to the severity of the disease. HR types are more common in cervical precancerous or malignant lesions, while LR types are more common in benign cases (4). There are approximately 50 million new cases of cervical cancer each year, with over 25 million cases of death expected. Thus, HPV infection and cervical cancer remain the major cancers threatening women worldwide (5). For this reason, HPV screening is suggested for the prevention of cervical cancer because it is more sensitive than cytological methods (6, 7).

Clinical studies have shown that 99.7% of cervical cancers are associated with HR-HPV infection. Although in most cases of infection, HPV may be cleared, persistent HR-HPV infection leads to severe cervical intraepithelial neoplasia (e.g., CIN I, CIN II or CIN III) and progression to cervical cancer (7, 8). Therefore, analytical methods for HPV viral DNA have been developed and marketed. The U.S. food and drug administration has approved the Hybrid capture2(HC2) system (Digene, Gaithersburg, Md., USA) and the AMPLICOR HPV test (Roche molecular diagnostics, Branchburg, N.J., USA). These screening tests determine the presence of common HPV (HR or LR strains) without discriminating between their genotypes. Although these tests provide good HPV screening tests, the inability to genotype HPV limits their use in clinical diagnosis and prognosis because the severity of disease caused by different strains varies greatly, with type 16 causing the most severe disease, followed by types 18, 33, 45, 59, and so on (8). Furthermore, persistent infection with HPV of the same genotype will further increase the risk of cervical cancer (9). Therefore, efficient and cost-effective HPV genotyping assays are most desirable.

LINEAR ARRAY HPV genotyping (Roche molecular diagnostics, Branchburg, N.J., USA, covering 37 genotypes) and INNO-LIPA HPV genotyping (Innogenetics, Belgium, covering 28 genotypes) can be used as a complement to HC2 or AMPLICOR HPV testing to distinguish specific HPV genotypes and determine infections involving multiple HPV genotypes. Based on the following facts: 1) the carcinogenicity of different HR-HPV strains varies; 2) persistent infection of the same genotype results in increased risk; 3) existing HPV vaccines do not prevent all types of HPV infection, and despite routine screening to determine the presence or absence of HPV virus, HPV genotyping is still desirable. However, the above-mentioned tests are very expensive and still use conventional hybridization processes, requiring high running costs and manual operation time. Most importantly, these genotyping kits do not detect more than a predetermined genotype profile and therefore produce a higher false negative rate in HPV screening. Thus, there is a need for HPV genotyping assays that are faster, cheaper, cover more genotypes, and are therefore more efficient to implement as an affordable alternative to current HPV detection.

Summary of The Invention

HLA genotyping

Preliminary results indicate that allele-specific oligonucleotide reverse dot blot (ASO-RDB) directed hybridization is a better choice when detecting DNA sequences of specific target HLA. The data obtained from this represent characteristic fragments at HLA-DP, DR and DQbeta sites, thus enabling accurate genotyping. The 83 DPB1 alleles determined by the World Health Organization (WHO) can be efficiently classified using a pair of PCR primers and 35 ASO oligonucleotide probes. Also, using the same pair of PCR primers and 18 ASO oligonucleotide probes, this simple hybridization method can identify the first two codes of a particular genotype at the DR and DQ beta sites, sufficient to distinguish these major human leukocyte antigens. ASO data were verified by direct sequencing. However, when the same PCR primer pair is used for direct sequencing of DR and DQ sites, some unexplained data is often generated because the same primer pair can also simultaneously amplify highly homologous endogenous pseudogene fragments within the HLA cluster. For this reason, DNA sequencing (a gold standard considered by many) may not guarantee HLA classification results. In such cases, to confirm ASO data, a number of sets of PCR primers corresponding to each HLA type to be tested are created and used in separate PCR reactions to obtain amplification products for sequencing. This is one of the main reasons why direct sequencing methods are costly and time consuming. In contrast, the present invention provides a low cost HLA identification procedure by performing a simple multiplex PCR using universal primer pairs and then hybridizing the required number of ASO probes on a low density spot array platform. The resulting HLA fragments (including pseudogenes) were amplified and were subjected to a definitive HLA classification on a membrane coated with an ASO probe. This is therefore a much superior approach to other current DNA or serological approaches. Although this straightforward flow-through approach requires additional oligonucleotide probes for further specific DR, DQ subtype classification, its number is well within the detection capabilities of this platform. The present invention provides a faster and simpler HLA typing technique that does not require expensive equipment and therefore is less costly to manufacture and use than direct DNA sequencing methods and multiplex PCR gel electrophoresis procedures.

The HLA genotyping primers and oligonucleotide probes disclosed herein have been tested and demonstrated to be useful in the above-described corresponding HLA-DR, DB and DP gene classification. More primers or oligonucleotide probes can be obtained, tested, and validated as shown in the design of FIG. 1 or FIG. 1A, allowing for more comprehensive genotyping. Although in the data validation example, a PCR reaction is used for amplification, other methods may be used as long as a sufficient number of specific target sequences are available for ASO-RDB flow through hybridization analysis. Other suitable amplification methods or techniques will be apparent to those of ordinary skill in the art upon reading the teachings herein. Amplification is not necessary as long as a sufficient number of specific target sequences are available for use in ASO-RDB flow-through hybridization assays. Detection may be accomplished by labeling the target DNA or the binder.

Although HLA genotyping is exemplified in this application, SNP-based genotyping techniques may also be applied to other genetic material or sequences obtained from any organism, such as the programs shown in fig. 1 or fig. 1A, following the teachings of this application. Devices like those described in U.S. patent nos. 5741547 or 6020187, or any new embodiment, capable of conducting flow-through hybridization may be used.

SNP genotyping

The human genome and the genomes of many other organisms have been sequenced and mapped. Within any species, general DNA sequence information is very similar. However, each species has its own unique set of genetic information. Thus, many scientists have attempted to find features in populations that are linked to disease. For example, anthropologists use genetic variation to reconstruct human history in an attempt to understand the effect of culture and geography on the global distribution of human trait populations. Single Nucleotide Polymorphism (SNP) data can accomplish these objectives (12). Brightwell et al reported the use of a SNP genotyping to analyze 6 SNP sites in a male population with FRAXA repeat expansion using a simple, rapid, single-tube, improved amplification-blocking mutation system (ARMS).

The present invention describes the use of Allele Specific Oligonucleotide (ASO) arrays. One of ordinary skill in the art, following the teachings of this application, can readily obtain the number of SNPs necessary to adequately identify a desired SNP. A membrane-based microarray ASO-RDB flow through hybridization platform (see, e.g., U.S. patent No. 5741647) can be used for SNP genotyping. The visible spots generated on the microarray hybridization platform of the present invention can be detected visually or analyzed using a low cost image analyzer. In contrast, hybridization platforms currently available on the market require analysis by high resolution image analyzers. In principle, single nucleotide polymorphisms at any position in the genome, provided that they are sufficient in number, can be used for identification purposes. However, this may affect the accuracy of paternity testing and genetic relationship analysis because of the differences in gene mutation rates in different parts of the genome. Thus, highly polymorphic sites or sites with relatively low mutation rates in the genome (including but not limited to coding regions or any regions with a relatively low mutation rate that satisfy the conditions) may be selected to verify relatedness. Preliminary data from single nucleotide polymorphism analyses performed on 9 highly polymorphic chromosomal sites show that these single nucleotide polymorphisms have been sufficient for SNP genotyping. The number of sites required will depend on the accuracy required for identification, as will be apparent to one of ordinary skill in the art upon reading the teachings herein. In constructing the polymorphic frequency database for each site, DNA samples taken from 50-150 unrelated individuals were sequenced. Analysis of 20 family relatives was performed in parallel with the STRProfile Plus human identity kit test with 100% agreement. SNP-based guided platforms have proven to be a better choice for human identification. In addition to the data that has been accumulated and analyzed, expansion of the polymorphic frequency database can be readily accomplished by one of ordinary skill in the art after reading the teachings herein.

SNP genotyping as a diagnostic tool

In addition to establishing DNA fingerprinting, SNP genotyping can also be used to identify gene fragments, or gene polymorphisms that cause alteration or attenuation of gene function. For this reason, the present invention can be used for rapid and definite identification of infectious agents, genetic diseases caused by specific DNA sequences, or genetic diseases caused by the presence or absence of such infectious agents or DNA sequences.

HPV genotyping identification

The present invention provides a method for HPV genotyping based on PCR assay, comprising co-amplifying a human gene as an internal control by membrane-based flow-through hybridization (U.S. Pat. No. 5741647) to measure the presence of target HPV integration in a sample. This method identified 33 High Risk (HR) and Low Risk (LR) HPV virus genotypes (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 66, 68, 70, 71, 72, 73, 81, 82, 84). In addition, novel universal probes have also been designed to capture HPV genotypes with HPV consensus sequences. Thus, in addition to the 33 genotypes included in the test panel, the universal probe captures at least 5 other HPV viral subtypes in clinical screening with satisfactory sensitivity and specificity and at a greatly reduced cost (10), providing a better choice for HPV genotyping.

Although in the data validation example, a PCR reaction is used for amplification, other methods may be used as long as a sufficient number of specific target sequences are available for ASO-RDB flow through hybridization analysis. Other suitable amplification methods or techniques will be apparent to those of ordinary skill in the art upon reading the teachings herein. Amplification is not necessary as long as a sufficient number of specific target sequences are available for use in ASO-RDB flow-through hybridization assays. Detection may be accomplished by labeling the target DNA or the binder.

Similar to the flow directing devices described in U.S. patent nos. 5741547 or 6020187, or any new embodiment, devices capable of conducting flow-directed hybridization processes may be used.

Drawings

FIG. 1 shows the method of the invention for constructing ASO probes and PCR primer libraries.

FIG. 1A shows a method of constructing an oligonucleotide probe library for HLA genotype identification according to the present invention.

FIG. 2 shows HLA-DQB genotype ASO detection results obtained using the method of the present invention.

FIG. 3 shows the results of ASO detection of HLA-DPB1 genotype using the method of the present invention.

FIG. 4 shows HLA-DRB and DQB genotype identification results for a sample.

FIG. 5 shows HLA-DPB genotype identification results for a sample.

FIG. 6 shows a membrane of the present invention for high throughput analysis.

FIG. 7 shows an exploded view of the hybridization device of the present invention, and a plurality of lateral flow guide detection devices connected to a central control unit. In one embodiment, the hybridization device comprises a central control unit, and one or more lateral flow devices connected to the central control unit. The central control unit provides power and controls the lateral flow device in which hybridization and development are performed. Multiple reactions (or multiple samples or analytes) can be tested simultaneously in a single lateral flow device, or can be tested simultaneously in several devices, each individually controlled under different conditions. The lateral flow device may be in an n x m lattice (array) format or a linear lattice format (as shown). Since the test solution flows from one end of the array to the other (i.e., from east to west or north to south) during the reaction, the sensitivity of the detection is significantly increased. The degree of increase in sensitivity depends on the ratio of the area of the membrane to the area of the spot or line containing the capture probe. For example, assuming a total area of 100 square millimeters, the spot size is 1 square millimeter. In a direct flow-through process (i.e., the solution flows from the top of the membrane to the bottom of the membrane as in a conventional flow process), only 1/100 of the test solution used will flow through this point, and only at this point will the target molecule bind to the probes immobilized on the membrane. However, if a lateral flow guiding process is used, the sensitivity depends only on the ratio of the width of the spot and the width of the membrane (e.g. the cross-section of the membrane). For example, in a lateral flow through process, on a 10 mm x 10 mm membrane, the volume of liquid flowing over a 1 mm wide spot will be about 1/10 of the total amount, which means a 10-fold increase in sensitivity using the same amount of target analyte (test solution containing the target molecule). When a linear array is used in the lateral flow-through process, the sensitivity will be further improved because all target molecules will pass through the lines distributed along the strip (or membrane). The lateral flow-through process allows quantitative measurements because the flow of the analyte during hybridization is more uniform.

FIG. 8 illustrates a method of the present invention for constructing a SNP database.

FIG. 9 shows data for identifying human or biological samples using SNP genotypes.

FIG. 10 shows the gene loci used in the method of FIG. 8.

FIG. 11 shows the result of HLA-DPB1 genotype identification in a sample of a Chinese population (Table 1).

FIG. 12 shows HLA-DPB1 allele and genotype frequencies in a sample of the Chinese population (Table 2).

FIG. 13 shows ASO oligonucleotide probe sequences for identifying HLA genotypes, and PCR primer pair sequences for amplifying corresponding fragments for analysis.

FIG. 14 shows the oligonucleotide probe sequences for identifying each HPV genotype, and the PCR primer pair sequences for amplifying the corresponding L1 fragment for analysis. "+ A" represents adenine-locked nucleic acid and "+ T" represents thymine-locked nucleic acid.

FIG. 15 shows a typical image of an HPV-33 genotyping identification array in HPV infection.

FIG. 16 shows a typical image of an HR-HPV-14 genotyping identification array in HPV infection.

FIG. 17 shows an improved HPV detection kit simplifying the screening format to reduce cost and increase throughput.

FIG. 18 shows other improved HPV detection kits to simplify screening formats to reduce cost and increase throughput.

Disclosure of Invention

The invention is described using the following terms. Unless specifically defined otherwise herein, the terms used to describe the present invention are intended to have a general meaning understood by those of ordinary skill in the art.

As used herein, "allele-specific oligonucleotide reverse dot blot (ASO-RDB)" refers to the immobilization of allele-specific oligonucleotide probes on a solid substrate surface for capture of the target molecule to be detected during hybridization.

As used herein, "flow-through hybridization" refers to a hybridization process using the technique described in U.S. Pat. No. 5741647.

As used herein, "flow directing hybridization device" or "flow directing device" refers to the apparatus described in U.S. Pat. No. 6020187 and/or the lateral flow device shown in FIG. 5, or any flow directing device designed subsequently.

HLA genotyping

The following text describes the method of the present invention for obtaining HLA typed ASO probes and PCR primers from genome sequence databases and for HLA genotyping analysis.

Selecting appropriate gene fragments and determining appropriate PCR primers and ASO sites

SNP or ASO suitable for HLA genotype identification is obtained by screening from GenBank or population screening by sequencing target genes or DNA fragments.

The method for selecting an appropriate target sequence in the present invention is described in detail below:

(a) data for all HLADNA sequences were found in GenBank according to the respective classification (class I, class II) and subtype (e.g., DR, DQ, DP, etc.). The HLA DNA sequences are aligned according to individual classification and subtype to determine the most polymorphic regions for HLA genotype identification.

(b) Within the polymorphic region, it was determined that the PCR primer pair could match the conserved sequences in the subtypes of interest, so that when these primers were used, all subtypes of interest could be amplified to give PCR products for further flow-through hybridization analysis.

(c) To verify the applicability of these primers, a large random sample was used for PCR amplification to obtain statistically satisfactory results for positive amplification of the region to be examined and to ensure that no alleles were excluded due to false negative amplification results. In addition to statistical confirmation, an Internal Control (IC) was also included in the validation to ensure that no PCR inhibitors were present in the test sample, resulting in failure of the PCR amplification reaction. The design of the internal control (which may be 1 or more) will depend on the test requirements. When genetic diseases are determined by means of HLA and SNP fingerprinting and genotyping, the sequence of the internal control cannot have homology with the human genome to be investigated, i.e.a sequence completely unrelated to the sequence to be investigated is used. Since human cells are mostly diploid genomes (except for germ line cells), the concentration problem is relatively simple, since theoretically the concentration of the test site is either 1 (homozygote) or 0.5 (heterozygote). Thus, two fragments with different sequences flanked by identical PCR primer sequences can be used as internal controls. This means that when a fragment different from the target fragment to be analyzed is used as an internal control, a sequence whose internal site is known to be homozygous or heterozygous can be selected as a probe, i.e., there are two different probes (pure and probe and heterozygous probe) in the fragment, so that the concentration ratio of these internal controls can be calculated. Since PCR amplification does not affect relative concentrations, the ratio of signals is theoretically 2: 1. Any fluctuation in the ratio, if any, will be seen as a change in hybridization efficiency. The two internal controls at a 2:1 ratio were used as standards to indicate that the sites to be examined were homozygote and heterozygote, respectively. If the observed results indicate only one allele, but at concentrations equal to or less than 0.5-fold of the homozygote internal control, or at ratios close to 1 to the heterozygote internal control, the possibility of loss of heterozygosity should be investigated: it may be that one of the two chromosomes is missing or cannot be amplified. Since the two chromosomes are identical, the expected result of imprinting points should be a concentration close to that of the homozygote internal control, rather than the heterozygote internal control. Internal controls are therefore very important to obtain accurate results, as clinically reliable results are crucial for any diagnostic test.

Further information on DNA databases and sequence alignments can be found in the references section of the present application (see references 5 and 6).

The procedures for HLA genotyping described herein may be used by those of ordinary skill in the art after learning the material of this application for genotyping other genes or DNA sequences of interest, or for SNP detection in conjunction with guided hybridization procedures to determine DNA fingerprinting/identification.

To maximize the efficiency of PCR amplification, the fragment length of the ASO probe is usually chosen to be as short as possible, preferably no more than a few hundred base pairs.

In the process of developing a genotype identification test, if a proper primer pair cannot be found, PCR reaction components and a PCR amplification process, namely a PCR program can be adjusted, and the amplification of a product is ensured by optimizing the amplification reaction conditions. Such modifications will become apparent to those skilled in the art upon a study of the present application. In some cases, if PCR primers with completely conserved sequences cannot be used, a degenerate primer or primers with the same recognition site can be used. In such cases, appropriate adjustments should be made to the PCR conditions to ensure that no sites are missed.

If no unique region can be found that successfully distinguishes between subtypes, multiplex PCR can be used. When designing the primers, the Tm value of the primers is determined to ensure that the annealing temperature during amplification is within an operable range. The "Tm value" herein refers to, for example, a reaction temperature at which the concentrations of double-stranded and single-stranded DNA molecules are equal. Thus, at higher temperatures more double stranded DNA dissociates into single strands, in contrast to lower temperatures where more single stranded molecules will anneal to form double strands. Population screening is usually performed by direct DNA sequencing for populations where no sequence data is known. For example, a sample of a Chinese population is screened as follows: (a) random sampling from more than one hundred subjects, PCR amplification using 2 pairs of primers, followed by hybridization using ASO probes, the probe design described in the following section; (b) and (5) DNA sequencing verification results.

Using the data obtained above, ASO sites used for genotyping were determined and selected. From the data obtained from GenBank or random sample sequencing, it was judged whether the site selection for HLA typing of the sampled population was indeed unique. The design and uniqueness verification method of the ASO probe is as follows:

(a) satisfactory polymorphic regions are selected from the sequence alignments of subtypes and further searches are made for unique 20-30 nucleotide sequences or fragments. Such unique sequences or fragments will be used as Allele Specific Oligonucleotide (ASO) probes to capture amplified target fragments by hybridization processes to detect the presence of each unique HLA subtype. By "unique sequence or fragment" is meant herein, for example, that among the subtypes of sequence, there is only one subtype of sequence that is completely homologous to the sequence or fragment.

(b) To verify that the ASO probes are unique, the sequences were aligned with all human DNA sequence data in GenBank or similar databases in europe to determine if the ASO probes did only match 100% of the HLA subtype of interest. This will ensure that the ASO probe is unique within the region, at least on the basis of existing data, until new sequences are found.

(c) Since the length of the PCR amplification product is short to improve amplification efficiency, a single ASO probe may not be sufficient to provide a clear conclusion to distinguish between distinct subtypes. Therefore, it is necessary to use multiple ASO probes to give a clear genotype classification in the same PCR fragment and/or in different fragments generated using multiplex PCR. In this case, a unique hybridization profile will be generated for each given HLA subtype. After extensive data analysis, ASO sequences were verified using random human DNA samples.

The lattice map of each genotype will be determined after the final determination of the number of ASO capture probes used. FIGS. 2 and 3 show examples of specific dot patterns. The dot-matrix maps shown in fig. 2 and 3 were used for determination of HLA DRB, DQB and DPB subtypes using 18 ASO probes and 35 ASO probes, respectively.

Similar procedures can be used for other genes and genetic material from other organisms. The primer sequences and number of ASO probes will vary for different genes and for different applications. For example, human identification may require 50 or more ASO lattices to obtain unambiguous identification (see fig. 3 and 4). The detection format may comprise an array of dots or lines, depending on the configuration of the flow-through hybridization device.

Performing ASO-RDB detection

The ASO oligonucleotides are immobilized on a membrane or any suitable substrate for capturing the target sites. By "membrane" is meant, for example, any suitable matrix material capable of immobilizing an ASO oligonucleotide probe and being porous to facilitate free passage of a solution containing the target nucleic acid molecule. In one embodiment, the membrane or matrix may be nylon, nitrocellulose, Biodyne, Porex, porous metal, or a durable gel matrix.

Immobilization of the target sequence (or site) may be achieved by covalent bonds, non-covalent bonds (i.e., electrostatic attraction, hydrophobic interactions, or other interactions including ultraviolet crosslinking), or by interaction through mediators, such as receptors or antibodies. Shown in FIGS. 2 and 3 is a Biodyne C membrane, with EDC used to form a covalent bond between the carboxy terminus of the membrane and the amino terminus of the modified ASO probe. Avidin-biotin linkages or poly-T tail uv cross-linking of ASOs are also effective.

The target sequence is amplified using appropriate primers to generate a sufficient amount of amplification product to satisfy the assay requirements. The target molecule may be appropriately labeled to generate a sufficient signal according to the final signal detection method. The labeling process may be carried out by primers, covalently attaching a labeling molecule to the 5' end of one or both primers, or by replacing one of the four dNTP nucleotides with a labeled nucleotide during PCR amplification, so that the newly extended amplification product is labeled. The label may be any molecule whose signal can be detected and developed. Biotin in combination with avidin labeled enzymes can be used for color detection. Other suitable labeling systems including, but not limited to, colloidal gold and fluorescent labels, magnetic bead conjugates, quantum dots, chemiluminescent labeling molecules, or other suitable systems that have been or are yet to be developed may also be employed.

ASO analysis was performed using the flow-through hybridization method described in U.S. patent No. 5741647. Hybridization is performed in a hybridization chamber with ASO capture probes cross-linked to the membrane. The ASO assay method is as follows:

(a) denaturing the solution containing the target DNA or sequence and contacting with a membrane;

(b) washing the membrane with a washing solution (or SSC, or blocking solution), preferably three times;

(c) developed color and detected visually or measured spectroscopically. For quantitative measurements, a scanner and image processing software can be used for analysis. Alternatively, the target DNA or sequence may be labeled with a fluorescent dye and analyzed using a spectral imager immediately after the washing step.

The results were compared to known sequence data to ensure accuracy of genotype detection.

The probes and test conditions were further optimized to improve the accuracy of genotype identification and to validate the RDB-ASO data by DNA sequencing.

Authentication

Verification was performed using random samples. One verification step of the present invention is described below:

once the capture probes of the ASO are immobilized on the membrane, a sufficient number of random DNA samples are used for PCR amplification, as described above. The target sequences or amplified and labeled DNA products/molecules are hybridized to generate an ASO lattice map.

HLA subtypes and corresponding DNA sequences were determined by ASO probes. Corresponding PCR samples were prepared for direct DNA sequencing. Verification of HLA genotype detection requires that the DNA sequencing results match those of the flow-through array. Samples for genotyping validation may be obtained from any randomly selected individual. Once a sample is obtained, true genotyping can be performed by DNA sequencing as described in section 3.5. If the sequencing data is consistent with the data obtained from the flow-through array, the validity of the data is confirmed. Further validation of genotype testing can be performed by field tests, such as random sampling tests, data analysis and statistical comparisons, such as sensitivity, specificity, positive predictive value or negative predictive value, all performed in independent laboratories to assess the accuracy of genotype testing.

SNP genotyping

The following are methods of the present invention for constructing SNP databases and developing SNP genotype tests.

Selection of SNP sites and determination of probability of exclusion

The term "excluding probability" as used herein refers to, for example, the accuracy of a method of distinguishing between humans or organisms. For example, an exclusion probability of 100 billion or 1000 parts per billion means: when a selected number of SNP sites are used, 100 or 1000 million individuals need to be screened to find two identical individuals, respectively.

SNP sites were selected and the probability of exclusion was determined. The term "excluding probability" as used herein refers to, for example, the accuracy of a method of distinguishing between humans or organisms. For example, an exclusion probability of 100 billion or 1000 parts per billion means: when a selected number of SNP sites are used, 100 or 1000 million individuals need to be screened to find two identical individuals, respectively.

The selection of appropriate SNP oligonucleotide probes to capture a particular target sequence to be analyzed is available either from screening data in GenBank or for population screening, such as: the DNA of the target gene or target DNA fragment is directly sequenced to obtain SNP information and frequency in the population.

From these data, the SNP sites to be used for fingerprint detection are determined according to polymorphism frequency, and it is determined whether the selected site is a mutation hotspot in this population, which can be determined by sequencing of random samples in the population. The number of SNP probes required was determined, and the total heterozygosity was calculated to determine the exclusion probability of the SNP site used for the analysis (i.e., SNP site array shown in FIG. 9).

The probability of exclusion depends on the number of SNPs at that site. For example, at a given site, if there are 2 different bases, such as G and A, each is found to account for 50% of the sample population. Such probability is 1/2. If 50 such loci could be found (which are randomly distributed and not linked to each other on the chromosome), then the probability of discrimination would be 1/2 to the power of 50.

Performing SNP map detection (SNP dot matrix combination shows the corresponding genotype composition of each individual site)

Appropriate primers are designed for amplification, and after screening and determination of sites as described above, appropriate SNP probes are selected for hybridization detection.

The target sequence was amplified and the SNP pattern was analyzed by flow-through hybridization as described in U.S. Pat. No. 5741647.

The data obtained above was compared with known sequence data to assess the accuracy of SNP genotyping assays.

Optimizing SNP probes and test conditions to improve the accuracy of SNP genotype identification analysis. RDB SNP data were verified by DNA sequencing.

Method validation was performed with random samples.

HPV genotyping identification

In one embodiment, the present invention provides a method for rapidly identifying Human Papilloma Virus (HPV) genotypes. The invention disclosed herein may also be applied to screening and detecting other viruses, bacteria, parasites or other pathogens. For example, the present invention may be used for genotyping tuberculosis, hepatitis b, hepatitis c, infectious atypical pneumonia and respiratory infection viruses (alone or in combination).

In one embodiment, a method for rapid detection of Human Papilloma Virus (HPV) comprises the steps of: (a) obtaining a sample comprising a nucleic acid template; (b) amplifying the template nucleic acid by using the primers of the sequence numbers 116-118 to obtain an amplification product of HPV L1; (c) the HPV L1 amplification product was hybridized to immobilized oligonucleotide probes selected from the group consisting of SEQ ID NO. 121-173, and the results of the hybridization indicated the presence of HPV viruses, including High Risk (HR) HPV viruses and Low Risk (LR) HPV viruses. In one embodiment, the primer contains a signal label. In another embodiment, the primer further comprises a primer complementary to the internal control (e.g., a primer comprising sequence No. 119-120). In yet another embodiment, the oligonucleotide probes further comprise a probe capable of capturing universal HPV viral DNA (e.g., a probe having the sequence of SEQ ID NO: 170-173), wherein said probe comprises a locked nucleic acid. In the present invention, use is made ofModified nucleotides are capable of increasing the thermostability and target specificity of oligonucleotide probes for HPV detection.

In one embodiment, the hybridization of the amplification product of HPV L1 to a plurality of immobilized oligonucleotide probes is performed in one or lateral flow through process. In one embodiment of the flow-through process, the sensitivity of hybridization is determined by the ratio of the area occupied by the probe to the total area of the membrane used for the array. In one embodiment of the lateral flow-through process, the sensitivity of hybridization depends on the ratio of the cross-sectional area occupied by the probe to the total cross-sectional area of the membrane in the direction of flow.

In one embodiment, the above method may detect one or more of the following HPV genotypes: HPVs 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 66, 68, 70, 71, 72, 73, 81, 82 and 84. HPV genotypes can be detected individually or grouped into one or more groups (e.g., see FIGS. 15-18). In one embodiment, HPV16 and 18 may be detected separately, while HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 73, 82 may be grouped into one or more groups for detection (see fig. 16-18), for example: HPV31, 33, 35, 39 may be detected as one group, HPV45, 51, 52, 56 as a second group, HPV58, 59, 73, 82 as a third group. In another embodiment, the method may further detect HPV6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 57, 61, 66, 70, 71, 72, 73, 81 and 84 in one or more groups (see example in fig. 18).

The invention also provides a kit for genotyping Human Papilloma Virus (HPV), which comprises the primer with the sequence number of 116-118 and the oligonucleotide probe selected from the sequence number of 121-173. In one embodiment, the primer further comprises a signal label. In another embodiment, the primer further comprises a primer complementary to the internal control (e.g., a primer comprising sequence numbers 119-120). In yet another embodiment, the oligonucleotide probes further comprise probes capable of capturing universal HPV viral DNA (e.g., probes having the sequence of SEQ ID NO: 170-. In one embodiment, the kit can detect one or more of the following HPV genotypes: HPVs 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 66, 68, 70, 71, 72, 73, 81, 82, and 84. HPV genotypes can be detected separately, or grouped into one or more groups for detection (see examples in FIGS. 15-18).

The invention will be more readily understood by reference to the following experimental details. Those skilled in the art will readily appreciate that the specific experimental details are for purposes of illustration only and are not intended to limit the invention to the details shown, which are set forth in the following description.

Various references or publications are cited in this application. The complete disclosures of these references or publications are incorporated herein by reference to more fully describe the state of the art to which this invention pertains. It is noted that the transitional word "comprising" is synonymous with "including", "containing" or "characterized by", is inclusive, open-ended, without limitation, and does not exclude additional, unrecited elements or method steps.

Example 1

Test program

DNA extraction

The following experimental protocol is recommended, but alternatives that are obvious and equally effective to a person skilled in the art may also be employed. Nucleated cells such as white blood cells or tissues are taken, washed with PBS, centrifuged, and the supernatant discarded. The cell pellet was resuspended in 200 microliters of PBS. DNA was extracted using the QIAamp DNA mini kit (QIAGEN) according to the manufacturer's recommended "blood and body fluid centrifugation protocol". Other commercially available kits for isolating DNA may also be used. However, the extraction process of DNA cannot dope the final purified DNA with DNA polymerase inhibitors, which is critical to ensure efficient amplification. The DNA was eluted in 50-200. mu.l of AE buffer and stored at-20 ℃ until use.

PCR amplification

Since PCR amplification is an extremely sensitive process, special care must be taken to prevent cross-contamination or false positive results. Therefore, the following criteria must be observed: in the PCR process, operating gloves are always worn, and a PCR reaction mixture is prepared in a front PCR area or a clean PCR preparation table without amplification products; the PCR reaction and hybridization should be performed in different regions, e.g., hybridization should be performed in the "post-PCR" region.

The bench or work area was cleaned using 70% ethanol and paper towels. All pipettes were cleaned with 70% ethanol and paper towel before starting PCR amplification. All pipetting steps used filtration/sterile tips, without repeated use of tips.

PCR is only one of many available techniques for amplification. Other amplification techniques, as would be apparent to one of ordinary skill in the art, may also be used, such as various methods of isothermal amplification, using appropriate primers to amplify the target sequence to obtain sufficient quantities of the target sequence (or DNA molecule) for flow-through array detection.

The PCR reaction uses commercially available polymerases such as:polymerase (applied biosystems). Five pairs of primers (1 pair for amplifying DR gene, i.e., forward primer DRB-F1: 5'-ATCCTTCGTGTCCCCACAGCACG-3' [ SEQ ID NO. 97]]And reverse primer DRB-R1: 5'-GCCGCTGCACTGTGAAGCTCTC-3' [ Serial number 98](ii) a 1 pair for amplifying the DQ genes, namely: forward primer DQB-E2-F2: 5'CGGTGATTCCCCGCAGAGGAT-3' [ SEQ ID NO. 99]And a reverse primerDQB-E2-R2: 5'-CCACCTCGTAGTTGTGTCTGC-3' [ Serial number 100](ii) a 3 pairs of forward primers for amplifying the DP gene [ SEQ ID NO. 101-]And a reverse primer [ sequence No. 104- ] 106]Biotin was labeled at each 5' -end. Other suitable marking methods as described above may also be used. PCR preliminary mix reagents were prepared in a 25. mu.l reaction system as follows (this example is intended to illustrate one particular procedure for the PCR amplification reaction of the invention. if primer sequences for other SNPs are used, PCR and hybridization conditions must be optimized):

	each reaction (microliter)	10 reactions
			PCR mixture	19.00	190.0
DNA polymerase	1 microliter (1 unit)	10 microliter (10 units)
			DNA template	5 (100 nanogram)	-
Total up to	25	200

The amplification procedure was optimized on a PE9700 thermal cycler:

9700PE (or MJ thermocycler):

5 minutes at 95 DEG C

95 ℃ for 20 seconds

30 seconds and 40 cycles at 55 DEG C

72 ℃ for 30 seconds

Final extension at 72 ℃ for 5 min

When different primers or other thermal cyclers are used, the cycling program may need to be modified.

PCR quality control

Positive and negative controls are important in each PCR analysis. A positive control is required to demonstrate PCR efficiency and specificity, and a negative control is used to determine whether any PCR reagents are contaminated. And an internal contrast is also needed to provide a correct interpretation to ensure the reliability and accuracy of the data. The type and number of internal controls depends on the type or nature of the test. Internal controls can be used to follow each step in the hybridization reaction or process. For example, an internal control can be used to determine whether a sample is added or whether an inhibitor is present in the sample, resulting in a PCR reaction that does not work properly. Internal controls may also be used to track the efficiency of the reaction, or to determine the concentration of the target molecule in a semi-quantitative or quantitative manner.

If quantitative or semi-quantitative measurements are desired, the detection array or membrane may have an internal control with signal markings on the membrane that provide a detection signal of a predetermined concentration when a predetermined program or predetermined conditions are met. In such embodiments, the internal control requires simultaneous development of color with the test sample after hybridization. In another embodiment, an additional internal control may be added to the PCR reaction mixture to indicate the efficiency of the PCR itself. This can also serve as an internal control, indicating the presence or absence of PCR reaction inhibitors.

The addition of an internal control system to the PCR reaction mixture of the present invention has several advantages: since the PCR reaction is carried out in the same reaction tube, the simultaneous absence of the internal control and test sample signals indicates the presence of the inhibitor; if the same primers are used for PCR amplification of the internal control and the test sample, the internal control can be used as an endogenous control to indicate the reaction efficiency of PCR itself; internal controls may also be used to determine the detection range (or cut-off value) of the PCR reaction and/or the efficiency of the hybridization process, and indicate whether the hybridization process was successfully completed. In addition, internal controls can be used to determine whether the signal developing reagent is properly or properly formulated, and whether the hybridization process is properly performed.

Hybridization of

Preparation before hybridization:

(1) prior to use, the hybridization solution (e.g., 2 XSSC or any commercially available solution/product) is preheated to 42 ℃ in a water bath. If there is a precipitate in solution B (SSC + 0.5% SDS), solution B is incubated at 42 ℃ until the precipitate dissolves. The temperature was maintained at 42 ℃ throughout the hybridization to maintain the set stringency.

(2) Preparing NBT/BCIP working solution: one tablet was dissolved in 10 ml of solution C or PBS buffer (phosphate buffered saline). The diluted NBT/BCIP working solution was stored protected from light and the unused solution was stored at 4 ℃.

(3) The hybridization solution (2 XSSC + 0.05% tween20) was equilibrated to room temperature.

(4) All biotin-labeled PCR products were denatured, heated at 95 ℃ for 5 minutes, then immediately cooled on ice for at least 2 minutes.

Preparation for hybridization

In flow-through hybridization assays, flow-through devices described in U.S. patent No. 6020187, or lateral flow devices described in this application, may be used. Switching on the power supply of the hybridization device, adding distilled water and preheating to 42 ℃.

The detection membrane coated with capture probes (e.g., as listed in the sequence listing below) is placed in a hybridization chamber. The membrane is secured, for example, with a lid.

Hybridization of PCR products

When the temperature reached 42 ℃ (+/-0.5 ℃), prehybridization was performed by adding 1 ml of preheated hybridization solution to cover the whole membrane. Incubate for at least 2 minutes and cover to prevent heat loss during prehybridization. Ensuring that the temperature equilibrates at the set point.

0.5 ml of the preheated hybridization solution was added to each denatured PCR product, and then the DNA sample was added to the designated well. The DNA sample containing the target sequence was brought into contact with the membrane surface, incubated at 42 ℃ for 5 minutes and then the DNA sample was allowed to flow over the membrane surface. Hybridization is usually completed within 30 seconds. The incubation time of 2-5 minutes is to ensure that the temperature of the DNA sample reaches the set temperature.

The membrane was washed 3 times with 0.8 ml of hybridization solution.

Color development

The temperature was set to 37 ℃. The pump was started and 0.5 ml of blocking solution was added immediately. The pump is stopped. An additional 0.5 ml of blocking solution was added, incubated for 5 minutes, and then all the solution was pumped out.

The pump was turned off and 0.5 ml of enzyme conjugate was added. The membrane was allowed to stand for 3 minutes. The color reaction works normally at 25-37 ℃. The pump was started and the membrane was washed thoroughly with 0.8 ml of buffered saline solution pH7.4 (preferably 4 times).

The pump was turned off and 0.5 ml NBT/BCIP solution (from Roche) was added.

The lid was closed and incubated for approximately 5 minutes or until color developed. Note that: the incubation time does not exceed 10 minutes.

The pump was started and the NBT/BCIP solution was pumped away. After the development has ended, the membrane is washed with 1 ml of solution B, preferably 3 to 4 times, and once with 2 ml of distilled water.

The results are preferably checked as soon as possible within not more than 1 hour, and may be detected semi-quantitatively by direct visual observation, or by scanning the image.

Interpretation of results

A positive result is indicated if a clearly visible spot appears in the color. 96 ASO probes (DRB29, DQB124 and DPB 143) in total correspond to sequences 1 to 96 in the sequence table of the HLA gene cluster, and 5 pairs of PCR primers correspond to sequences 97 to 106 in the sequence table for amplification. The design of the probes and primers is shown in FIGS. 1 and 1A. These ASO probe and primer pairs have been evaluated and determined to be appropriate for classification of HLA DR, DQ and DP genes. FIG. 2 shows typical results of analyzing HLA-DRB and DQB sites using 18 ASO probes, respectively, and typing according to a dot-matrix labeling pattern of genotypes. Similarly, FIG. 3 shows the results of HLA-DPB 1. For this gene, 35 of 43 designed ASO probes were selected for generating a dot-matrix map (32 probes were finally used after random sample screening and validation). A summary of the ASO-HLA DR, DQ, and DP data is shown in FIGS. 4 and 5. The genotypes and allele frequencies are given in tables 1 and 2. Data obtained from reverse phase spot array flow-through hybridization experimentsA total of 141 random human samples were used-confirmed by DNA sequencing individually. In principle, any known ASO (or SNP) oligonucleotide from an organism, if sufficient data is available for genetic analysis, can be genotyped using the rapid flow-through hybridization method described herein.

The results of hybridization are available within a few minutes, meaning that the detection speed is at least 10 times greater than conventional hybridization techniques.

Example 2

Simplified genotyping protocol and apparatus

Methods and apparatus for flow-through hybridization of DNA, as described in U.S. Pat. Nos. 5741647 and 6020187, respectively, reduce hybridization time from hours or days to minutes (the entire hybridization assay can be completed in 5-30 minutes, depending on the signal detection method used). The device is inexpensive to manufacture and consumes 10 times less reagents than conventional hybridization devices, and thus it would be a more affordable DNA diagnostic technique.

The present invention provides an inexpensive platform for studying the interaction of nucleic acids, proteins and other compounds using low density arrays. As described above, the genotyping method of the present invention is a significant improvement over conventional hybridization processes.

The present invention also improves the existing flow-through hybridization technique. For example, the hybridization device of the present invention comprises a detection membrane, as shown in FIG. 6 in a 4X 6 lattice format. If an ELISA96 well plate format is used, each well is prepared as a 5-point array, and 96 samples and 5 different assays can be processed simultaneously. This greatly improves the throughput of the assay.

The format of the lattice can be modified by one of ordinary skill in the art to add more wells for rapid and cost-effective analysis of a larger set of nucleotide sequences. The hybridization device of the invention is a breakthrough of DNA rapid diagnosis. Figure 7 shows a lateral flow and micro model example of the device of the present invention.

Significant improvements in hybridization protocols have also been published, including:

(1) the prehybridization step is eliminated, and a modified reagent mixture is used to allow simultaneous blocking and hybridization (i.e., without prehybridization, the DNA sample is placed in hybridization solution in direct contact with the flow-through detection membrane)

(2) In the one-step hybridization method, a target sequence or molecule is marked with a fluorescent label, a quantum dot, a colloidal gold particle, a magnetic bead or other suitable labels, so that the step of enzyme-linked substrate color development is not required. These modifications allow the technician to complete the entire hybridization process in 5 minutes or less. The method of the invention can further save time and reagent consumption.

Example 3

Single Nucleotide Polymorphism (SNP) based genotyping

From 50 to 400 individual samples, 8 gene clusters and 55 gene fragments were selected and sequenced to determine appropriate sites for SNP genotyping. Figure 9 shows one of the combinations of sites used for fingerprinting. The results were compared with the STRProfiler Plus fingerprint kit (Applied Biosystems) to ensure accuracy. Fig. 10 shows a locus used in the fingerprinting method of fig. 8. Probes and primers for other candidate genes or sequences can be readily determined by one of ordinary skill in the art based on the teachings of the present application. Genes that have been tested include globin genes, BRCAs, apolipoprotein E, collagen, p53, G6PD deficiency genes and HLA DP, DQ and DR, which cause thalassemia. Any SNP known to an organism, if sufficient data is available for genetic analysis, can be tested or detected using the rapid SNP typing method of the present invention.

In identifying DRB genotypes, primer pair DRB-F1 was used in PCR: 5'-ATCCTTCGTGTCCCCACAGCACG-3' [ sequence number: 97] and DRB-R1: 5'-GCCGCTGCACTGTGAAGCTCTC-3' [ SEQ ID NO: 98], and 29 ASO probes were tested, 18 of which were found to be most suitable for identifying HLA-DRB alleles. In identifying the DQB1 genotype, PCR reactions were performed using the primer pair DQB-E2-F2: 5'-CGGTGATTCCCCGCAGAGGAT-3' [ sequence number: 99] and DQB-E2-R2: 5'-CCACCTCGTAGTTGTGTCTGC-3' [ sequence number: 100], the reaction can generate a 260bp fragment. 24 SSO probes were used as capture probes for this DQB1 class hybridization.

In identifying the DPB1 genotype, a total of 43 ASO probes were tested, of which a set of 35 SSO probes was shown as an example. To obtain hybridization detectable levels of product from amplification of a target gene or sequence, a set of primers is used to perform multiplex PCR amplification. Primer1-f, Primer2-f and Primer3-f as forward primers and Primer4-r, Primer5-r and Primer6-r as reverse primers. These primer pairs were able to generate approximately 264bp, 5' -end-labeled amplicons for hybridization and development to determine the genotype to be tested.

Example 4

HPV genotyping identification

FIG. 14 shows an oligonucleotide probe for capturing a specific HPV L1 region (SEQ ID NO: 121-. To ensure that the PCR reaction proceeded normally and that no inhibitor was present in the reaction, a pair of internal control primers were also added (SEQ ID NO: 119 and 120) and co-amplified with the HPV viral genome. In addition, a biotin-labeled oligonucleotide HC was also immobilized to each dot matrix as a control in color development.

The HPV genotyping is performed by using the flow guide membrane array platform, the operation is quick and simple, and the flow guide device is provided with a replaceable separator, so that the lattice format can be easily changed to adapt to different lattice forms. The use of the reaction chamber can also be optimized by adjusting the size and number of the spots, achieving maximum cost savings. In one example, the HPV33 genotyping kit currently uses a 5 × 6 array, which allows 15 assays per run. In another embodiment, the 4 × 5 lattice can be extended to measure 30 samples per run. The 2 x 3 array is an ideal choice for high throughput screening because it can run 64 arrays simultaneously for validation assays using the same size equipment as the ELISA plate, a very effective and affordable alternative method for screening HPV infections. In yet another embodiment, the dot matrix membrane may also be designed for use in the micro-hybridization apparatus shown in FIG. 7.

FIG. 15A shows the detection of a typical 5X 6 array, which clearly shows a single type of HPV infection. Multiple infections are shown in figure 15B. The 4 x 5 dot matrix shown in figure 16 was used to screen 14 HR-HPV genotypes recommended in a cancer prevention program. Fig. 17 shows a 2 × 3 lattice format. The HPV detection kit shown in fig. 17A serves three purposes: (a) definitive identification of whether to infect HPV type 16 or 18, or both, covers approximately 70% of HPV infections. Knowing this result, enables the clinician to make the correct diagnosis, prognosis and treatment, including decision to vaccinate despite the presence of HPV infection; (b) the HR-HPV point covers a group of 12 HR-HPV types, provides clinical detection data, and is the most effective method for preventing cervical cancer widely recommended by worldwide organizations (such as the world health organization); (c) the universal point is used for universal HPV screening, and not only can 14 cancer-causing HR-HPV be detected, but also other HPV of intermediate types, lower-risk or unknown types can be detected, so that the universal point can be used for early screening for preventing HPV. Furthermore, this can be repeated as another internal to ensure that the test is validated, as distinguished from all tests currently on the market. Therefore, despite the low cost, this new HPV kit will be the most comprehensive tool for HPV and cervical cancer screening and prevention.

PCR amplification, signal display, result interpretation and validation

PCR amplification, hybridization, color development and interpretation of results are similar to those already described above, except that the primers of SEQ ID NO. 116-118 are used, and a higher concentration of Tag polymerase is used to compensate for the insufficient activity of the degenerate primers. It will be apparent to those skilled in the art that image signals may be generated by other color rendering techniques and that quantitative results may be obtained by image scanning techniques. However, due to sample differences, absolute quantitative results for similar assays are neither achievable nor necessary; visual inspection is sufficient because primary screening for the presence or absence of the virus for HPV is sufficient and, most importantly, the cost of detection is affordable. In the present invention, the L1 region of HPV is used for the determination. The forward primer (HPVF) was Biotin-5'-GCMCAGGGWCATAAYAATGG-3' (SEQ ID NO: 116). For amplification enhancement, two reverse primers, i.e., HPVR Biotin-5'-CGTCCMARRGGAWACTGATC-3' (SEQ ID NO: 117) and HPVR25'-GCGACCCAATGCAAATTGGT-3' (SEQ ID NO: 118), were used. The amplification procedure was optimized on a PE9700 thermal cycler, and the following procedure was verified using primers (SEQ ID NO: 116-118):

9700PE (or MJ thermocycler):

5 minutes at 95 DEG C

95 ℃ for 20 seconds

42 cycles at 55 ℃ for 30 seconds

72 ℃ for 30 seconds

Final extension at 72 ℃ for 5 min

When different primers or other thermal cyclers are used, one skilled in the art can readily modify the cycling procedure to achieve optimal amplification efficiency.

Reference to the literature

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Claims (4)

1. A kit for genotyping Human Papillomavirus (HPV), which is characterized by comprising a primer with a sequence number of 116-118 and oligonucleotide probes with a sequence number of 123-125, 127-133, 140-145, 148-149, 151-153, 156-158, 170-173, wherein the probes with a sequence number of 123-125, 127-133, 140-145, 148-149, 151-153 and 156-158 correspond to the HPV L1 nucleic acid sequences with genotypes of HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68, and the probes with a sequence number of 170-173 correspond to the consensus sequence of the HPV L1 nucleic acid.

2. The kit of claim 1, further comprising a primer complementary to the internal control.

3. The kit according to claim 2, wherein the primer complementary to the internal control has a sequence number of 119-120.

4. The kit of claim 1, wherein the primer comprises a signal generating label.