CN105907878A - Drug resistance gene mutation detection of tuberculous bacillus based on high-resolution melting curve technology - Google Patents

Abstract

The invention relates to the detection of drug-resistant gene mutations of mycobacterium tuberculosis (TB) based on high-resolution melting curve technology. Specifically, the present invention relates to a PCR primer set for detecting drug-resistant gene mutations of Mycobacterium tuberculosis (TB) based on high-resolution melting curve technology. The invention also relates to a kit and a microarray for detecting drug-resistant gene mutations of Mycobacterium tuberculosis (TB) based on a high-resolution melting curve technique comprising a PCR primer set. The invention also relates to the use of the PCR primer set in preparing a kit and a microarray for detecting drug-resistant gene mutations of Mycobacterium tuberculosis (TB) based on high-resolution melting curve technology. The invention also relates to a method for detecting the drug resistance gene mutation of Mycobacterium tuberculosis (TB) in a sample by using the PCR primer set.

Description

Drug-resistant gene mutations in Mycobacterium tuberculosis based on high-resolution melting curve technology detection

technical field

The invention relates to the detection of drug-resistant gene mutations of Mycobacterium tuberculosis (TB) based on high-resolution melting curve technology. Specifically, the present invention relates to a PCR primer set for detecting drug-resistant gene mutations of Mycobacterium tuberculosis (TB) based on high-resolution melting curve technology. The invention also relates to a kit and a microarray for detecting drug-resistant gene mutations of Mycobacterium tuberculosis (TB) based on a high-resolution melting curve technique comprising a PCR primer set. The invention also relates to the use of the PCR primer set in preparing a kit and a microarray for detecting drug-resistant gene mutations of Mycobacterium tuberculosis (TB) based on high-resolution melting curve technology. The invention also relates to a method for detecting the drug resistance gene mutation of Mycobacterium tuberculosis (TB) in a sample by using the PCR primer set.

Background technique

High-resolution melting curve (high-resolution melt, HRM) analysis technology is an efficient and robust PCR technology, not limited by the site and type of mutation base, without sequence-specific probes, run high-resolution melting directly after PCR , to complete the analysis of sample mutation and single nucleotide polymorphism-SNP. Its main principle is to analyze the sample according to the length of DNA sequence, GC content and base complementarity difference, and its high temperature uniformity and temperature resolution make the resolution accuracy reach that of a single base. Differentiation of base differences. HRM uses the characteristic that specific dyes can be inserted into DNA double strands, and records the high-resolution melting curve by real-time monitoring of the combination of double-stranded DNA fluorescent dyes and PCR amplification products during the heating process, so as to detect samples. For example, in the detection of SNP, the unmatched double-stranded DNA at the SNP site will be untied first during the heating process, and the fluorescent dye will be released from the partially unzipped DNA molecule, and the existence of SNP can be judged from the fluorescence intensity and time curve , and different SNP sites, heterozygosity or not will affect the peak shape of the melting curve, so HRM analysis can effectively distinguish different SNP sites and different genotypes.

Clinically, the commonly used first-line drugs for tuberculosis caused by Mycobacterium tuberculosis include isoniazid (INH), rifampicin (RFP), streptomycin (SM) and so on. The mechanism of action of isoniazid is that it is activated by catalase-peroxidase in Mycobacterium tuberculosis, acts on enoyl carrier protein reductase, inhibits the biosynthesis of mycobacterial cell wall and kills bacteria. The katG gene is a gene encoding catalase-peroxidase, and its gene mutation can lead to a decrease or even loss of catalase-peroxidase activity. Mutations in the katG gene account for about 50-70% of isoniazid resistance. InhA gene encodes reduced nicotinamide dinucleotide (NADH)-dependent enoyl reductase, and its gene mutation accounts for about 10-35% of isoniazid resistance.

Rifampicin is a broad-spectrum antibacterial drug that can bind to the β subunit of DNA-dependent RNA polymerase to inhibit the activity of RNA polymerase, thereby inhibiting mRNA transcription and protein synthesis to play an antibacterial role. About 95% of rifampicin-resistant bacteria are due to mutations in the gene encoding RNA polymerase beta subunit (rpoB), which no longer binds to rifampicin and produces drug resistance. The rpoB gene mutation mainly occurred in the 81bp region encoding 27 amino acid codons at positions 507-533.

A variety of biological detection techniques have been applied to the detection of drug resistance of Mycobacterium tuberculosis, such as phage biological amplification method, conventional drug sensitivity test, DNA sequencing, gene chip, PCR-restriction fragment length polymorphism analysis, rapid culture Instrument detection system, etc.

The phage biological amplification method and conventional drug susceptibility testing are cumbersome and time-consuming, and difficult to standardize. The preparation and labeling of gene chip samples are cumbersome, the instruments are expensive, and the detection cost is high. PCR-restriction fragment length polymorphism analysis can only analyze gene mutations at specific loci with known sequences. Rapid culture instrument detection system instruments and reagents rely on high import costs. At the same time, some non-sequencing methods need to rely on enzyme digestion or clustering to interpret the gene mutation status of patients, and there is a greater probability of typing errors. Although the accuracy of direct sequencing is high, the DNA fragments generated by the sequencing reaction need to be separated by capillary electrophoresis, and then the fluorescent signal is detected by the detection system, which is time-consuming and expensive.

The HRM technology is simple and quick to operate, low in cost of use, and accurate in results, realizing true closed-tube operation. Therefore, the application and promotion of HRM technology in the detection of Mycobacterium tuberculosis resistance genes (inhA, katG, rpoB) will have great value and potential.

Contents of the invention

One aspect of the present invention relates to a PCR primer set for detecting drug resistance gene mutations of Mycobacterium tuberculosis (TB) based on high-resolution melting curve technology, the PCR primer set includes at least one primer set selected from the following:

(1) primer set 1, it comprises the primer with the nucleotide sequence shown in SEQ ID NO:1 and the primer with the nucleotide sequence shown in SEQ ID NO:2;

(2) primer set 2, which comprises a primer with a nucleotide sequence shown in SEQ ID NO: 3 and a primer with a nucleotide sequence shown in SEQ ID NO: 4; and

(3) Primer set 3, which comprises a primer having a nucleotide sequence shown in SEQ ID NO: 5 and a primer having a nucleotide sequence shown in SEQ ID NO: 6.

In one embodiment, the PCR primer set includes primer set 1, and optionally at least one primer set selected from primer set 2 and primer set 3. In another embodiment, the PCR primer set includes primer set 2, and optionally at least one primer set selected from primer set 1 and primer set 3. In yet another embodiment, the PCR primer set includes primer set 3, and optionally at least one primer set selected from primer set 1 and primer set 2. In yet another embodiment, the PCR primer set includes primer set 1, primer set 2, and primer set 3.

Another aspect of the present invention relates to a kit or microarray for detecting drug-resistant gene mutations of Mycobacterium tuberculosis based on high-resolution melting curve technology, which comprises the PCR primer set of the present invention. Another aspect of the present invention relates to the use of the PCR primer set of the present invention in the preparation of a kit or microarray for the detection of Mycobacterium tuberculosis drug resistance gene mutations in samples based on high-resolution melting curve technology.

In one embodiment, said sample is a biological sample, preferably said sample is a body fluid sample or a tissue sample, and more preferably said sample is selected from a biopsy sample, a cell culture, a solidified sample (such as a paraffin-packed buried samples), whole blood, plasma, serum, saliva, cerebrospinal fluid, sweat, sputum, alveolar lavage fluid, urine, feces, secretions, breast milk, and peritoneal fluid.

Another aspect of the present invention relates to a method for detecting the drug-resistant gene mutation of Mycobacterium tuberculosis in a sample based on high-resolution melting curve technology, the method comprising:

(1) extracting and optionally purifying nucleic acids in a sample,

(2) PCR amplification using the primer set of the present invention, and

(3) Carry out high-resolution melting curve analysis to the PCR product of step (2);

Wherein the method is not used for diagnostic purposes, for example, it can be used to detect whether Mycobacterium tuberculosis in in vitro culture is mutated, and can be used to detect whether Mycobacterium tuberculosis samples from environmental sources have drug resistance. .

In one embodiment, said sample is a biological sample, preferably said sample is a body fluid sample or a tissue sample, and more preferably said sample is selected from a biopsy sample, a cell culture, a solidified sample (such as a paraffin-packed buried samples), whole blood, plasma, serum, saliva, cerebrospinal fluid, sweat, sputum, alveolar lavage fluid, urine, feces, secretions, breast milk, and peritoneal fluid. In another embodiment, the conditions of the high-resolution melting curve analysis are: 95°C for 2 minutes, scanning from 83°C to 96°C or scanning from 82°C to 93°C or scanning from 80°C to 94°C at 0.1°C/step. In one embodiment, in one embodiment, the PCR amplification can be a qualitative or quantitative PCR amplification. In another embodiment, the PCR amplification is quantitative PCR amplification. In yet another embodiment, the PCR amplification is real-time fluorescent quantitative PCR amplification.

Description of drawings

Figure 1 is a screenshot of the result of detecting the mutation of the rpoB gene by high-resolution melting curve analysis after using the PCR primer set of the present invention (primer set 3, SEQ ID NO: 5 and 6) to amplify the rpoB gene.

Fig. 2 is a screenshot of the result of detecting the mutation of the katG gene by high-resolution melting curve analysis after using the PCR primer set of the present invention (primer set 2, SEQ ID NO: 3 and 4) to amplify the katG gene.

Fig. 3 is a screenshot of the result of detecting the mutation of the inhA gene by high-resolution melting curve analysis after using the PCR primer set of the present invention (primer set 1, SEQ ID NO: 1 and 2) to amplify the inhA gene.

Figure 4 is a screenshot of rpoB gene amplification using different PCR primer sets. A: A PCR primer set (SEQ ID NO: 8 and 6) with the same reverse primer as the PCR primer set 3 of the present invention but with a small portion of different bases on the forward primer. The results show that NTC has severe non-specific amplification (Fig. 4A gray curve); B: PCR primer set of the present invention (primer set 3, SEQ ID NO: 5 and 6), the results show that there is no non-specific amplification.

detailed description

Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a thorough understanding of the invention. One of ordinary skill in the relevant art will readily recognize, however, that the invention may be practiced without one or more of the specific details or may be practiced otherwise.

The purpose of the present invention is to solve the shortcomings of the detection of the drug-resistant gene mutation of Mycobacterium tuberculosis (TB) in the prior art, such as large typing errors, long time-consuming or high detection cost. The present invention solves the above-mentioned problems by using the high-resolution melting curve technology of the PCR primer set of the present invention.

The high-resolution melting curve technique using the PCR primer set of the present invention has the following advantages:

1) The instrument and reagents can achieve the best detection effect through system optimization, and it is easy to standardize;

2) The test results are automatically given by the software to avoid the subjectivity of the result judgment;

3) The operation process is simple and time-saving. The whole detection process takes 120 minutes, including 100 minutes for automatic operation of the instrument and 20 minutes for manual operation; and

4) There is no non-specific amplification and the detection accuracy is high.

Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Definitions of general terms in cell and molecular biology can be found in: Gene VIII translated by Yu Long et al., Standard Book Number: ISBN:978-7-03-014597-0, published by Science Press (2005); translated by Zhang Jinfeng et al. Cell and Molecular Biology, published by CITIC Press (2004), ISBN: 978-7-50-860075-8; Biochemistry edited by Wang Jingyan, published by Higher Education Press (2002), ISBN: 978-7-04 -011088-3; Kendrew, J. et al. (eds), The Encyclopedia of Molecular Biology, Blackwell Science Ltd. Published (1994), ISBN 0-632-02182-9; and Meyers, R.A. (eds), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc. (1995), ISBN 1-56081-5698. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the specific materials and methods are described herein.

sample

The term "sample" as used herein includes any sample containing nucleic acid molecules. Samples may be derived from biological sources ("biological samples"), such as tissues (e.g., biopsy samples), extracts or cultures including Mycobacterium tuberculosis, and biological or physiological fluids, such as solidified samples (e.g., paraffin-packed Buried samples), whole blood, plasma, serum, saliva, cerebrospinal fluid, sweat, sputum, alveolar lavage fluid, urine, feces, excretion, milk, peritoneal fluid, etc. Samples obtained from sources or after pretreatment to improve sample characteristics (eg, preparation of plasma from blood, dilution of sputum, etc.) can be used directly. In certain aspects of the invention, the sample is sputum or alveolar lavage fluid.

Samples that may be analyzed and/or used in accordance with the present invention include polynucleotides of clinical origin, such as DNA or RNA.

The method of extracting nucleic acid from a sample is well known in the art, such as DNA extraction with phenol and chloroform, or using commercially available DNA extraction reagents. For example, extraction can be performed using a column kit (eg, GENERATION (registered trademark) Capture Column Kit Gentra).

It should be understood that nucleic acids can be purified by conventional purification methods in the art, such as using the PrepSEQ™ kit (from Applied Biosystems) and the methods in US Pat. No. 5,234,809, among others.

Drug resistance, also known as drug resistance, refers to the tolerance of microorganisms, parasites and tumor cells to the effects of chemotherapy drugs. In the present invention, drug resistance refers to the drug resistance of Mycobacterium tuberculosis, especially refers to the drug resistance of Mycobacterium tuberculosis to the first-line drugs (including isoniazid (INH), Rifampicin (RFP), streptomycin (SM), etc.) drug resistance. Drug resistance gene mutations in some embodiments of the present invention refer to mutations in the genes inhA, katG and/or rpoB of Mycobacterium tuberculosis.

Primer

A "primer" as used herein generally refers to a linear oligonucleotide that is complementary to and anneals to a target sequence. The lower limit on primer length is based on hybridization ability, since very short primers (eg, less than 5 nucleotides) do not form thermodynamically stable duplexes under most hybridization conditions. Primers typically vary in length from 8-50 nucleotides. In certain embodiments, primers are between about 15-25 nucleotides. As used herein, the term "forward primer" refers to an oligonucleotide that anneals to a specific strand of target DNA. As used herein, the term "reverse primer" refers to an oligonucleotide that anneals to the opposite strand of the target DNA. In summary, the forward and reverse primers are generally oriented on the target DNA sequence in a manner similar to PCR primers such that their 3' ends are closer to the target sequence than their 5' ends. Naturally occurring nucleotides (especially guanine, adenine, cytosine and thymine, hereinafter referred to as "G", "A", "C" and "T") and nucleotide analogs, are useful primers of the present invention. As used herein, the term "PCR primer" refers to an oligonucleotide primer used to initiate a PCR reaction on a nucleic acid.

As used herein, "PCR product" refers to an amplified nucleic acid produced from a nucleic acid template by nucleic acid PCR amplification.

As used herein, the term "nucleotide analog" refers to compounds that are structurally similar to naturally occurring nucleotides. Nucleotide analogs can have altered phosphate backbones, sugar moieties, nucleobases, or combinations thereof. Nucleotide analogs, often with altered nucleobases, among others, confer different base-pairing and base-stacking properties. Nucleotide analogues with altered phosphate-sugar backbones (eg peptide nucleic acid (PNA), locked nucleic acid (LNA)) typically alter chain properties, eg secondary structure formation, among other things.

Examples of PCR primer sets and target genes used to detect drug resistance gene mutations in Mycobacterium tuberculosis (TB) based on high-resolution melting curve technology are shown in Table 1.

Table 1 Primer sets used in the present invention and corresponding target genes

The nucleotide sequence of the PCR primer of the present invention also includes its modified form, as long as the amplification or HRM analysis effect of the primer is not significantly affected. The modification can be, for example, adding one or more nucleotide residues in or at both ends of the nucleotide sequence, deleting one or more nucleotide residues in the nucleotide sequence, or changing one or more nucleotide residues in the sequence Multiple nucleotide residues are substituted for additional nucleotide residues, eg, A is replaced by T, C is replaced by G, etc. It is clear to those skilled in the art that the modified primers are also covered by the present invention, especially the protection scope of the claims. In one embodiment, the modified form of the nucleotide sequence of the PCR primer is a chemically enhanced primer as disclosed in CN103270174A.

Each nucleotide in the primer of the present invention can be chemically synthesized using, for example, a general-purpose DNA synthesizer (eg, Model 394 manufactured by Applied Biosystems). Oligonucleotides can also be synthesized by any other method well known in the art, such as PCR primers.

Genomic DNA extracted from the sample was used as a template, and PCR primers were used to amplify the drug-resistant gene mutation of Mycobacterium tuberculosis (TB) to obtain amplification products. Amplification reactions include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCP), self-sustaining sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA) , Multiple Displacement Amplification (MDA) and Rolling Circle Amplification (RCA), which are disclosed in the following references (hereby incorporated by reference): Mullis et al., U.S. Patent Nos. 4,683,195; 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et al., U.S. Patent No. 5,210,015 (Real-time PCR with "Taqman" or "Taq" [registered trademark] probes); Wittwer et al., U.S. Patent No. 6,174,670; Kacian et al., U.S. Patent No. 5,399,491 ("NASBA"); Lizardi, US Patent No. 5,854,033; Aono et al., Japanese Patent Publication No. JP 4-262799 (Rolling Circle Amplification); and others.

The target nucleotides are preferably amplified using the PCR method. The PCR method itself is well known in the art. The term "PCR" includes derivatives of this reaction including, but not limited to, reverse transcription PCR, real-time PCR, nested PCR, multiplex PCR, and quantitative PCR, among others. Preferably, the quantitative amplification of the target nucleotide is carried out by using the fluorescent quantitative PCR method. In one embodiment, in one embodiment, the PCR method can be a qualitative or quantitative PCR method. In another embodiment, the PCR method is a quantitative PCR method. In yet another embodiment, the PCR method is a real-time fluorescent quantitative PCR method.

Using a primer that hybridizes to the sense strand (reverse primer) and a primer that hybridizes to the antisense strand (forward primer) in the presence of primers, template DNA, and thermostable DNA polymerase, the denaturation, annealing, and extension steps The cycle is repeated about 30 to 50 times (for example, 45 times) to perform PCR. In one embodiment, the PCR is real-time PCR. In one embodiment, PCR is performed using saturating fluorescent dyes (eg, LC Green, LC Green Plus, Eva Green, and SYTO-9). In one embodiment PCR uses primer sets 1, 2 and/or 3 and any combination thereof (for example the combination of primer sets 1 and 2 (when the target genes are inhA and katG), the primer sets 1 and 2 and 3 combination (when the target genes are inhA, katG and rpoB, etc.). The PCR primer set of the present invention can be combined arbitrarily to detect different target genes in the sample, for example, primer set 1 can be combined with primer set 2 and/or 3, primer set 2 can be combined with primer set 1 and/or 3, Primer set 3 can be combined with primer sets 1 and/or 2. Therefore, in one embodiment, the PCR primer set of the present invention may comprise primer set 1, and optionally at least one primer set selected from primer set 2 and primer set 3. In another embodiment, the PCR primer set of the present invention includes primer set 2, and optionally at least one primer set selected from primer set 1 and primer set 3. In yet another embodiment, the PCR primer set of the present invention includes primer set 3, and optionally at least one primer set selected from primer set 1 and primer set 2.

In the PCR of the present invention, various conventional thermostable DNA polymerases can be used for amplification, including but not limited to FastStart Taq DNA polymerase (Roche), Ex Taq (registered trademark, Takara), Z-Taq, AccuPrime TaqDNA polymerase and HS Taq DNA polymerase.

The method of selecting suitable PCR reaction conditions based on the Tm value of the primers is well known in the art, and those skilled in the art can select the optimal conditions according to the primer length, GC content, target specificity and sensitivity, the nature of the polymerase used, etc. For example, a PCR reaction can be performed using the following conditions: 95°C for 5 minutes, 95°C for 15 seconds, 65°C for 10 seconds, 72°C for 10 seconds, 45 cycles.

After the PCR product is obtained, the mutation site of the amplified target gene is detected according to the high-resolution melting curve method. In one embodiment, the conditions for high-resolution melting curve analysis are: 95°C for 2 minutes, scanning from 83°C to 96°C (for the rpoB gene) or scanning from 82°C to 93°C (for the katG gene) at 0.1°C/step Or scan from 80°C to 94°C (for the inhA gene). In one embodiment, high resolution melting curve analysis is performed using a Rotor-gene Q PCR instrument.

Reagent test kit

The present invention also relates to a kit for detecting drug-resistant gene mutations of Mycobacterium tuberculosis (TB) based on high-resolution melting curve technology, which contains the PCR primer set of the present invention. The present invention also relates to the use of the PCR primer set of the present invention in preparing a kit for detecting drug-resistant gene mutations of Mycobacterium tuberculosis (TB) based on high-resolution melting curve technology. In one embodiment, the PCR primer set is at least one (eg at least 2 or all 3) primer sets selected from primer set 1, primer set 2 and primer set 3. In one embodiment, the PCR primer set of the present invention may comprise primer set 1, and optionally at least one primer set selected from primer set 2 and primer set 3. In another embodiment, the PCR primer set of the present invention includes primer set 2, and optionally at least one primer set selected from primer set 1 and primer set 3. In yet another embodiment, the PCR primer set of the present invention includes primer set 3, and optionally at least one primer set selected from primer set 1 and primer set 2.

Kits may contain materials or reagents (including PCR primer sets) useful in practicing the methods of the invention. Kits may include storage of reaction reagents (eg, primers, dNTPs, enzymes, etc.) and/or supporting materials (eg, buffers, instructions for performing the assay, etc.) in suitable containers. For example, a kit can include one or more containers (eg, boxes) containing corresponding reaction reagents and/or supporting materials. Such contents may be delivered together or separately to the intended recipient. For example, a first container may contain an enzyme for the assay, a second container a saturating fluorescent dye (eg, LC Green, LC Green Plus, EvaGreen, and SYTO-9), and a third container a PCR primer set. The kit may also contain a compartment suitable for containing the reagents or containers. As an example, a kit may contain a PCR primer set, a saturating fluorescent dye (such as LC Green, LCGreen Plus, Eva Green, and SYTO-9), a PCR reaction buffer, and an instruction manual. The kit can also contain polymerase, dTNP and the like. The kit can also contain UNG, internal standards for quality control, positive and negative controls, etc. The kit may also contain reagents for preparing nucleic acid, such as DNA, from a sample. The kit of the present invention may also contain any other PCR primer sets other than the PCR primer set of the present invention, for example, a PCR primer set capable of effectively detecting drug resistance gene mutations of Mycobacterium tuberculosis (TB). The above examples are not to be construed as limiting the kits and their contents suitable for use in the present invention.

microarray

The present invention also relates to a microarray for detecting drug-resistant gene mutations of Mycobacterium tuberculosis (TB) based on high-resolution melting curve technology, which contains the PCR primer set of the present invention. The present invention also relates to the use of the PCR primer set of the present invention in preparing a microarray for detecting drug-resistant gene mutations of Mycobacterium tuberculosis (TB) based on high-resolution melting curve technology. In one embodiment, the PCR primer set is at least one (eg at least 2 or all 3) primer sets selected from primer set 1, primer set 2 and primer set 3. In one embodiment, the PCR primer set of the present invention may comprise primer set 1, and optionally at least one primer set selected from primer set 2 and primer set 3. In another embodiment, the PCR primer set of the present invention includes primer set 2, and optionally at least one primer set selected from primer set 1 and primer set 3. In yet another embodiment, the PCR primer set of the present invention includes primer set 3, and optionally at least one primer set selected from primer set 1 and primer set 2.

Microarray refers to a solid support with a flat surface that has an array of nucleic acids, each member of the array comprising identical copies of oligonucleotides or polynucleotides immobilized on spatially defined regions or sites, so The regions or locations do not overlap with regions or locations of other members of the array; that is, the regions or locations are spatially discrete. Furthermore, spatially defined hybridization sites may be "addressable" in that their locations and the identities of the oligonucleotides to which they are immobilized are known or predetermined (e.g., prior to their use). definite). Typically the oligonucleotide or polynucleotide is single stranded and is covalently linked to the solid support, usually at the 5'- or 3'-end. The density of nucleic acids containing non-overlapping regions in a microarray is typically greater than 100/cm ² , more preferably greater than 1000/cm ² . Microarray technology is disclosed, for example, in the following references: Microarrays: A Practical Approach edited by Schena (IRL Press, Oxford, 2000); Southern, Current Opin. Chem. Biol., 2:404-410, 1998, the entire contents of which are accessed by incorporated herein by reference.

While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Many changes to the disclosed embodiments may be made in accordance with the disclosure herein without departing from the spirit or scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above-described embodiments.

All documents mentioned herein are hereby incorporated by reference. All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document was indicated individually.

Example

Unless otherwise stated, the materials used in the examples herein are all commercially available, and the various specific experimental methods used to carry out the experiments are all routine experimental methods in the art (see for example "Refined Molecular Biology" edited by F. Osper et al. "Guide to Molecular Cloning Experiments" (1999), Science Press, ISBN 7-03-006408-9 and "Molecular Cloning Experiment Guide (Third Edition)" (2002), Science Press, ISBN 7 -03-010338-6) or according to the steps and conditions suggested by the manufacturer, and can be routinely determined by those skilled in the art as needed. Certain materials and methods are detailed below.

Example 1: High-resolution melting curve analysis using the PCR primer set of the present invention

1. Materials and Methods

1.1. Materials

PCR reaction solution 1 (including PCR primer set 1), PCR reaction solution 2 (including PCR primer set 2), PCR reaction solution 3 (including PCR primer set 3) and HS Taq enzyme.

The concentration of each component in the PCR reaction solution is shown in Table 2.

Table 2

name Final concentration source forward primer F 0.3uM Invitrogen reverse primer R 0.3uM Invitrogen dNTP 0.2mM Roche Fluorescent dye Eva Green 1x Biotium 20x buffer 1x QIAGEN MgCl ₂ 2mM PG

1.2. Samples

Wild-type control plasmid wt (inhA-katG-rpoB gene wild-type plasmid (SEQ ID NO: 7), synthesized by Invitrogen Company) and hospital clinical sputum sample nucleic acid.

1.3. Equipment

QIAGEN Rotor-gene Q PCR instrument.

1.4. Method

Firstly, the purified genomic DNA of Mycobacterium tuberculosis was amplified on the Rotor-Gene Q platform using PCR reaction solution and HS Taq. The PCR reaction solution contains fluorescent dyes, which can embed increasing PCR double-stranded products during the amplification process. middle. Therefore, the entire process of product enrichment can be monitored in real time by the software of Rotor-Gene Q, thereby ensuring reliable quality PCR products for subsequent HRM analysis. The running results are automatically analyzed by the software, which reduces the burden and error of manual analysis.

According to the required number of PCR reaction tubes n (n≤36), the reaction system is prepared as shown in Table 3:

table 3

PCR reaction solution HS Taq enzyme The reaction solution n×27.6 µL n×0.4µL

rpoB detection program:

● 95℃: 5 minutes;

● 95°C: 15 seconds, 65°C: 10 seconds, 72°C: 10 seconds, the fluorescence signal collection is set at 65°C, the signal collection channel is "Green", 45 cycles;

● HRM: 95°C: 2 minutes

83°C to 96°C (0.1°C/step)

katG detection program:

● 95℃: 5 minutes;

● 95°C: 15 seconds, 65°C: 10 seconds, 72°C: 10 seconds, the fluorescence signal collection is set at 65°C, the signal collection channel is "Green", 45 cycles;

● HRM: 95°C: 2 minutes

82°C to 93°C (0.1°C/step)

inhA detection procedure:

● 95℃: 5 minutes;

● 95°C: 15 seconds, 65°C: 10 seconds, 72°C: 10 seconds, the fluorescence signal collection is set at 65°C, the signal collection channel is "Green", 45 cycles;

● HRM: 95°C: 2 minutes

80°C to 94°C (0.1°C/step).

2. Results and Analysis

Through the analysis of the two analysis modes, if the sample can be clearly distinguished from the wild-type control, it is judged that the sample has a mutation in the gene detection region, otherwise it is judged as the wild type. The results are shown in Figure 1-3. Figure 1 shows a screenshot of the results of detecting mutations by high-resolution melting curve analysis after the rpoB gene is amplified using the PCR primer set (SEQ ID NO: 5 and 6) of the present invention. Figure 2 shows a screenshot of the results of mutation detection using high-resolution melting curve analysis after the katG gene is amplified using the PCR primer set (SEQ ID NO: 3 and 4) of the present invention. Fig. 3 shows a screenshot of the results of mutation detection by high-resolution melting curve analysis after the inhA gene is amplified using the PCR primer set (SEQ ID NO: 1 and 2) of the present invention.

As can be seen from Figures 1-3, the application of the PCR primer set of the present invention can quickly and accurately detect the mutations in the drug resistance gene mutations (inhA, katG and rpoB) of Mycobacterium tuberculosis, and can well distinguish between wild-type controls and heterozygous mutations and homozygous mutations, and were consistent with the detection results of sputum samples or DNA sequencing results (results not shown) of routine clinical drug susceptibility tests.

Embodiment 2: Comparison between different PCR primer sets

The materials, instruments, methods and conditions used in this example are the same as in Example 1, but in the PCR process, two PCR primer sets shown in Table 4 below were used to amplify the wild-type plasmid rpoB gene.

Table 4

The PCR primer set 3 (the primer set of the present invention) and the PCR primer set 4 are completely identical in the reverse primer, and only a small part of bases are different in the forward primer. The quality of the detection results of the same wild-type plasmid sample using different PCR primer sets (3 and 4) is completely different, see Figure 4. The results of PCR primer set 4 showed severe non-specific amplification of NTC (gray curve in Figure 4A), while there was no non-specific amplification in PCR primer set 3.

Claims (10)

1. be used for detecting the PCR primer set of mycobacterium tuberculosis resistance gene mutation based on high-resolution melting curve technology, said PCR primer set comprises at least one primer set selected from following: (1) primer set 1, it comprises the primer with the nucleotide sequence shown in SEQ ID NO:1 and the primer with the nucleotide sequence shown in SEQ ID NO:2; (2) primer set 2, which comprises a primer with a nucleotide sequence shown in SEQ ID NO: 3 and a primer with a nucleotide sequence shown in SEQ ID NO: 4; and (3) Primer set 3, which comprises a primer having a nucleotide sequence shown in SEQ ID NO: 5 and a primer having a nucleotide sequence shown in SEQ ID NO: 6. 2. The PCR primer set of claim 1, wherein the PCR primer set comprises primer set 1 and optionally at least one primer set selected from primer set 2 and primer set 3. 3. The PCR primer set of claim 1, wherein the PCR primer set comprises primer set 2, and optionally at least one primer set selected from primer set 1 and primer set 3. 4. The PCR primer set of claim 1, wherein the PCR primer set comprises primer set 3, and optionally comprises at least one primer set selected from primer set 1 and primer set 2. 5. A test kit or microarray for detecting the drug resistance gene mutation of Mycobacterium tuberculosis based on high-resolution melting curve technology, comprising the PCR primer set according to any one of claims 1-4. 6. Use of the PCR primer set according to any one of claims 1-4 in the preparation of a test kit or microarray for detecting the drug-resistant gene mutation of Mycobacterium tuberculosis in a sample based on high-resolution melting curve technology. 7. The purposes of claim 6, wherein said sample is a biological sample, preferably said sample is a body fluid sample or a tissue sample, and more preferably said sample is selected from the group consisting of biopsy samples, cell cultures, solidified samples ( Such as paraffin-embedded samples), whole blood, plasma, serum, saliva, cerebrospinal fluid, sweat, sputum, alveolar lavage fluid, urine, feces, secretions, breast milk, and peritoneal fluid. 8. A method based on high-resolution melting curve technology detection of Mycobacterium tuberculosis drug resistance gene mutation in a sample, said method comprising: (1) extracting and optionally purifying nucleic acids in a sample, (2) PCR amplification using the primer set of any one of claims 1-4, and (3) Carry out high-resolution melting curve analysis to the PCR product of step (2); The methods described therein are not intended for diagnostic purposes. 9. The method of claim 8, wherein said sample is a biological sample, preferably said sample is a body fluid sample or a tissue sample, and more preferably said sample is selected from the group consisting of biopsy samples, cell cultures, solidified samples ( Such as paraffin-embedded samples), whole blood, plasma, serum, saliva, cerebrospinal fluid, sweat, sputum, alveolar lavage fluid, urine, feces, secretions, breast milk, and peritoneal fluid. 10. The method according to claim 8 or 9, wherein the conditions for high-resolution melting curve analysis are: 95°C for 2 minutes, scanning from 83°C to 96°C or scanning from 82°C to 93°C or scanning from 80°C at 0.1°C/step to 94°C.