WO2005038427A2

WO2005038427A2 - Rapid identification of viruses of the upper respiratory tract infection including sars causing coronus viruses

Info

Publication number: WO2005038427A2
Application number: PCT/US2004/017767
Authority: WO
Inventors: Joseph Wing On Tam; Hok Sin Woo; Wing Sze Lo; Sin Fun Sia; Tsz Kam Fang
Original assignee: MEDICAL GENE CENTER Ltd
Current assignee: MEDICAL GENE CENTER Ltd
Priority date: 2003-06-05
Filing date: 2004-06-04
Publication date: 2005-04-28
Anticipated expiration: 2005-12-05
Also published as: WO2005038427A3

Abstract

This invention provides a method for the detection of a pathogen through analysis of nucleic acid sequences, comprising the steps of: selecting at least one appropriate target sequence from the pathogen; screening said sequence to obtain at least one appropriate primer for amplification; selecting an appropriate probe from the sequence; immobilizing the allelic-Specific-Oligonucleotide site of each sequence onto a solid matrix suitable for capturing the target sequence; amplifying the sequence(s) wherein the steps (d) or (e) can be performing concurrently or sequentially; performing blot analyses on each sequence; and comparing the results of the blot analysis with using positive or negative controls. This invention further provides a method for amplifying the target sequence of a pathogen for hybridization comprising the steps of: selecting an appropriate primer pair; performing reverse transcription on the primer pair; and performing the first and second round of PCR in a single tube.

Description

RAPID IDENTIFICATION OF VIRUSES OF THE UPPER RESPIRATORY TRACT INFECTION INCLUDING SARS CAUSING CORONUS VIRUSES

This is a continuation-in-part application Of U.S. Serial No. 60/476, 907, filed June 5, 2003, the content of which is incorporated into this application by reference in its entirety.

Throughout this application, various publications are referenced. Disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains .

FIELD OF INVENTION

The present invention relates to method of making definitive identification of upper respiratory viruses and Severe Acute Respiratory Syndrome (SARS) causing viruses such as coronus and related viruses in human and animals by DNA analysis and the device thereof.

BACKGROUND OF THE INVENTION

The Severe Acute Respiratory Syndrome (SARS) epidemic has drastically impacted the world recently, and different or new strains, genotypes or species of coronus viruses have been identified from patients with SARS. Subsequently, it has become obvious that the SARS virus has a very high rate of mutation, particularly when ribavirin or other antiviral drugs are used for treating the patients. Coronaviridae has been known and isolated from many other species including human. Although different strains of coronus virus may cause different severity of SARS, however, not all strains or genotypes of coronus virus, such as the Human coronavirus 229E, have been implicated for causing SARS. Therefore, simply detecting the existence or presence of the coronus virus without knowing the detailed characteristics, such as the genotypes or variants, is insufficient or inadequate for the diagnosis of SARS. Even within the subclass of the coronus virus isolated from SARS patients, different variants, which caused the disease in different severity, were identified. Hence, it is imperative that when detecting the coronavirus or related viruses we must not only detect the general type within a common region but also provide an additional region (s) for verification, or better yet, the device or method deployed in the field, such as the method and device of the present invention, should be able to identify the respective genotype (s) of the given virus .

Since other viruses or pathogens may also be responsible for causing SARS, simultaneous detection of such virus (es) or pathogen (s) is imperative to save lives . In the case of SARS, other than the confirmed pathogen of coronus virus, other pathogen, such as Chlamydia, myxoviruses, and C. pneumoniae, have also been reported to be the possible causative pathogens for SARS. Although it is still not definitive whether any of these latter pathogens are indeed the causative agents for SARS, the fact that they have been identified in patients with SARS means that the detection of these pathogens may be important in prescribing an effective treatment. For effective management and prevention of the spread of SARS, it is extremely important to be able to differentiate the patient (s) who are infected with other viruses which cause SARS-like symptoms, such as upper respiratory track infections, from the SARS patients as early as possible. One of the reasons that the death rates in some SARS-affected regions are unusually high is the indiscriminate use of drugs to treat patients who display SARS-like symptoms but, in fact, the symptoms or illnesses of those patients are not caused by the same causative pathogens, such as coronus virus; thus, rendering the treatments ineffective. For this and other health reasons, more accurate, comprehensive early detection method or device for the causative pathogens is urgently needed.

The present invention is based on a low-density array membrane flow-through system which provides a better detection format . Using the flow-through hybridization device, the present invention not only provides a fast, simple format for identifying the coronus virus with adequate controls, but also provides simultaneous identification of the known genotypes of the causative pathogens for SARS. Moreover, if needed, additional pathogens, whether SARS related or unrelated, can alsobedetectedinthe same reaction and/or in the same membrane or matrixes simultaneously. Once the results are known, appropriate and potentially life-saving treatment regimen specific to the identified pathoge (s) can be prescribed for the patient. Hence, this invention provides a novel method of analysis for SARS which can also be used for the analyses of other gene(s) or pathogens . The method is based on our patented principle of Direct Flow-through DNA Hybridization (Tarn JWO, US Pat. No. 5,471,547 (1998) & 6,020, 187 (2000) ) .

Direct Flow-through DNA Hybridization is the fastest annealing process that uses a very inexpensive device for accurate mutation detection, genotyping and fingerprinting analysis. The present invention, as applied and described herein, presents the scheme and data obtained from analyzing the SARS pathogen and SARS causative pathogens by RT-PCR and Allele (or gene or sequence) Specific Oligonucleotides (ASO) probes (oligo-probes) using the Flow-through format . This invention also presents an improved protocol or testing procedures to improve the detection sensitivity for the analyses and controls .

SUMMARY OF THE INVENTION

In accordance with these and other objects of the invention, a brief summary of the present invention is presented. Some simplifications and omission may be made in the following summary, which is intended to highlight and introduce some aspects of the present invention, but not to limit its scope. Detailed descriptions of a preferred exemplary embodiment adequate to allow those of ordinary skill in the art to make and use the invention concepts will follow in later sections .

Results suggested that the Allelic-Specific-Oligonucleotide Reversed-Dot- Blotting (ASO-RDB) direct Flow-through Hybridization is a good alternative for the detection of specific target pathogen (s) through the analysis of nucleic acids (DNA or RNA) sequences. Data obtained refer to the specific segments of coronus virus at multiple regions that are able to provide accurate determination of the genotypes and/or multiple species of pathogens, such as the upper respiratory track infectious viruses, using the fast hybridization procedures, and the ASO oligo-probes in a low-density array format of the present invention.

PCR amplification using multiple pairs of PCR primers (multiplex PCR) was used. Nucleic acids amplification can be performed using other amplification methods, such as strand displacement amplification (SDA) , transcription mediated amplification (TMA) , loop-mediated amplification (LAMP) and other amplification methods already established or new amplification technology to be invented in the future. The primers and oligo-probes shown in the sequence listing described herein and attached have been tested and confirmed to be useful for the upper respiratory viruses including SARS causing viruses. But these primers and oligo-probes are used as examples and, therefore, are not the only primers and oligo-probes which can be used with this invention; other primers and oligo-probes can be created by the methods set out in this invention as soon as the sequences of the viruses are known.

Using the scheme presented in Fig. 1, additional primers and oligo-probes can be selected, tested and validated for full genotyping in the future . Although PCR was used as the amplification method, any other method that can produce specific target sequences in enough quantity and/or without amplification if naturally occurring in high enough quantity for testing can also be used for the ASO-RDB flow through Hybridization determination. The detection can be by labeling of the target DNA or conjugates through any known or those developed in the future appropriate for present method of detection.

Although coronus virus and its variants and causative viruses of the upper respiratory track are being used as the practical example here, this SNP-based analysis method can be applied to other gene and sequences of any organism by following the general procedures as shown in Figure 1. The Flow-through Device shown in Figure 2 is one example. Hybridization devices, such as those described in U.S . Patents No. 5,741,547 or 6, 020, 187 or any new embodiments which are capable of carrying out the above-described flow-through hybridization process, can also be used.

DETAILED DESCRIPTION OF THE DRAWINGS

The drawings which illustrate specific embodiments of the invention should not be construed as restricting the spirit or scope of the invention in any way:

Fig. 1 is the diagram of the method for obtaining the ASO probes and PCR primers data base of the invention.

Fig. 2 is one of the examples the hybridization device already in production and registered elsewhere.

Fig. 3 is the agarose gel image showing the detection limit of SARS coronavirus down to 2 copies.

Fig. 4 shows the result of SARS coronus virus detection by flow-through hybridization. Positive signals are represented by blue dots on the pre-probed membrane.

Fig. 5 is the arrangement of the ASO probes of the membrane array.

Fig. 6 is the hybridization result of the SARS coronus virus variant (genotyping identification) detection. Table below shows three known strains or variants CA, CB, Tor (as the wide type) , and the mutated sequence probes that were used to test the correspondent mutants. The probe arrangement is given along with resulted image. SNP position for SARS coronavirus genotyping

Fig. 7 is the arrangement of the viral detection array for the upper respiratory track infection. Legend : 1. Respiratory syncytial virus (RSV) 2. Enterovirus (Entero) 3. Adenovirus (Adeno) 4. Parainfluenza virus type 1 (PIV1) 5. Parainfluenza virus type 3 (PIV 3) 6. Parainfluenza virus type 3B (PIV 3B) 7. Influenza A (Inf A) 8. Influenza B (Inf B) 9. SARS coronavirus (SARS) 10. Metapneumovirus (Meta) 11. Mycoplasma pneumoniae (M. Pneu) 12. Chlamydia pneumoniae (CP)

Fig. 8 shows the hybridization results of viral detection array after multiplex PCR: A) SARS coronavirus; B) Influenza A virus; C) Parainfluenza type 1 virus; D) Respiratory syncytical virus. The test specificity was evaluated with theuseofnucleicacid samples and probes of 10 other respiratory tract pathogens (respiratory syncytial virus (RSV) , parainfluenza virus type 1 & 3 (PIV 1 & 3) , Mycoplasma pneumoniae, Chlamydia pneumoniae, enterovirus, influenza A & B, adenovirus, metapneumovirus). No cross-reaction was detected. This figure shows examples of the specific HybriMax-based hybridization of SARS coronavirus and other pathogens PCR products to SARS coronavirus probe and other specific probes respectively.

Membrane positions of probes are as follows:

1. Respiratory syncytial virus (RSV)

2. Enterovirus (Entero)

3. Adenovirus (Adeno) 4. Parainfluenza virus type 1 (PIV1)

5. Parainfluenza virus type 3 (PIV 3)

6. Parainfluenza virus type 3B (PIV 3B)

7. Influenza A (Inf A)

8. Influenza B (Inf B) 9. SARS coronavirus (SARS)

10. Metapneumovirus (Meta)

11. Mycoplasma pneumoniae (M. Pneu)

12. Chlamydia pneumoniae (CP)

Analytical sensitivity was determined by RT-PCR of serial dilution of in vitro transcribed cloned RT-PCRproducts . The detection limits were as follows :

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method for the detection of a pathogen through analysis of nucleic acid sequences, comprising the steps of: (a) selecting at least one appropriate target sequence from the pathogen; (b) screening said sequence to obtain at least one appropriate primer for amplification; (c) selecting an appropriate probe from the sequence; (d) immobilizing the sequence-specific-oligonucleotide site of each sequence onto a solid matrix suitable for capturing the target sequence; (e) amplifying the sequence (s) wherein the steps (d) and (e) are performed separately;

(f) performing blot analyses on each sequence; and (g) comparing the results of the blot analysis with using positive or negative controls. In an embodiment, steps (d) and (e) are performed sequentially or concurrently. In another embodiment, the sequence is a gene, pathogen or allelic sequence. In an embodiment, the solidmatri isa low-density array for detecting multiple SARS variant. In another embodiment, the solid matrix is a viral detection array for detecting upper respiratory tract infection. In a further embodiment, the array is on a single membrane .

The pathogen includes but is not limited to the upper respiratory viruses. In an embodiment, the pathogen is a coronavirus. In another embodiment, the pathogen is an avian influenza virus. In another embodiment, the primer and the sequence-specific-oligonucleotide site are obtained by screening data from the GenBank or by sequencing the target sequence or in combination thereof. In a further embodiment, target sequence comprises a gene, nucleic acid or DNA sequence.

In a further embodiment, the probe is a single nucleotide polymorphism probe. In a further embodiment, the solid matrix is a membrane or nylon. In a further embodiment, a target sequence having a concentration less than 0.5 f moles is amplified by the above describe method. In further embodiment, the amplification step can be performed by PCR amplification, isothermal amplification, nested RT-PCR amplification, two-step nested RT-PCR amplification.

In a further embodiment, the upper respiratory viruses include but is not limited to a group selected from respiratory syncytial virus (RSV) , parainfluenza virus type 1 (PIV1) , parainfluenza virus type

3 (PIV3), parainfluenza virus type 3B (PIV3B) , Mycoplasma pneumoniae (MP) , Chlamydia pneumoniae (CP) , Enterovirus (EN), Influenza A (Inf A), Influenza B ( Inf B) ; Adenovirus (Adeno) , Metapneumovirus (Meta) , and SARS coronavirus (SARS) .

The invention provides a method for the detection of more than one pathogen through analysis of nucleic acid sequences, comprising the steps as described above. However, more than one pair of primers can also be used. In an embodiment, the detection procedure can be completed within two hours . In another embodiment, the detection procedure is finished within minutes . In a further embodiment, the detection procedure is finished within 20 minutes using enzyme link color development. Preferably, the detection procedure can be completed within between 2 to 3 minutes with using use colloidal gold or florescence dye.

This invention further provides a method for the detection of pathogens including but not limited to SARS causative pathogens.

This invention further provides novel polymerase chain reaction (PCR) primers and allele-, gene- or sequence-specific oligonucleotides

(ASO) probes or oligo-probes which can be used for detecting pathogens .

This invention further provides a method for identifying novel probes and primers which can be used to detecting pathogen (s) . This invention provides a kit for detection of pathogen comprising a compartment containing the primer (s) or probe (s) or a combination thereof, which are disclosed herein or developed by the methods as describe above. This invention provides a machine capable of detecting pathogens through analysis of nucleic acid sequences. In an embodiment, the machine can be programmed to automatically carryout the methods as described above.

This invention provides a method for amplifying the target sequence of a pathogen for hybridization comprising the steps of: selecting an appropriate primer pair; performing reverse transcription on the primer pair; and performing the first and second round of PCR in a single tube . In an embodiment, the second round of PCR is performed with a thermal cycling program having the following thermal cycling profile: (1) 95°C, 5 min; (2) 95°C, 10 sec; (3) 56°C, lOsec; (4) 72°C, 30sec; (5) 39 cycle repeats on steps 2 to 4, followed by 72°C, 10 min final extension time.

In another embodiment, the second round PCR was done using lul of the 1:10 dilution of the first round product in 50ul reaction volume . In a further embodiment, the reverse transcription is performed with positive control RNA of about 2xl0^"5 copies using the Thermoscript RT-PCR system and Platinum Taq DNA polymerase. In a further embodiment, thermal cycling profile: (1) 95°C - 3min; (2) 95°C , lOsec; (3) 60°C, lOsec (drop by l°C/cycle) ; (4) 72°C, 30 sec; (5) 9 cycle repeats on steps 2 to 4; (6) 95°C, lOsec; (7) 56°C, lOsec; (8) 72°C, 30sec; (9) 9 cycle repeats on steps 6 to 8; and (10) Store at 4°C. The following is one of the general procedures for the present invention:

(a) Select gene segments and determine the PCR primers and the ASO sites: 1. Select the appropriate target sequences to be analyzed by either screening data from the GenBank and/or by sequencing the target genes or target DNA segments to get the SNP (or ASO) profile related to the pathogen (s) . 2. From these data, determine the ASO sites to be used for genotyping based on the sequence data to evaluate if the sites are indeed unique for the genotype or variant within target species to be tested. 3. Determine the number of ASO capture probes.

(b) Perform ASO-RDB detection for genotyping: 1. Immobilizing the ASO oligonucleotides onto the membrane or any matrix for capturing the target loci. 2. Amplifying the target segments using the appropriate primers by labeling the amplified target DNA molecules for the ASO-probes for hybridization detection. 3. Performing ASO profile analyses by Flow-through Hybridization process using a device as depicted in figure The hybridization is done as described in pages 9-15 of the DNA HybriMax™ Owner' s Manual (Hong Kong DNA Limited) of the device. The following protocol is an example: i) Denature and drop the target DNA solution onto the membrane; ii) Wash and develop the color for visual inspection or spectrometric measurements (The target DNA can be labeled with fluorescence dye in which case direct spectrometric determination other than color development by enzyme linked immuno-specific assay is needed. Other developing methods like colloidal Gold or magnetic bead or quantum-dot or any other conjungate labeling systems already used or developed in the future can also be used) . iii) Results are compared with known sequence data for accuracy evaluation, iv) Modify the probes and testing conditions for accuracy. The RDB-ASO data are verified by DNA sequencing.

(c) Validation (for diagnostic kits development)

Proceed with validation using (i) control panels; (ii) random patient samples and; (iii) clinical trials.

Development scheme:

Following the scheme as in Figure 1 1. Select conserved region (s) for generic types (detection the existence of a given pathogens or genes, without detailed genetic/sequence specific) .

2. Put in Positive and negative controls to ensure the validation of the test

3. Select the specific regions ( for genotyping for a given species and/or different species)

4. Design the respective primers and detection probes for the test . 5. Establish complete protocols.

EXAMPLES

In examples which are intended to illustrate embodiments of the invention but which are not intended to limit the scope of the invention :

(I) Diagnosis for the SARS causative pathogen, the coronus virus and its variants

Detailed sequences of the first isolated coronus virus and subsequently the first 7 variants were submitted to the GeneBank for public knowledge. We designed the ASO probes of all 7 variants for the coronus viruses and four PCR primers for the amplification of the pathogen(s). These sequences are listed in the Appendix. In principle, the ASO or SNP probe sequences and specific primers sequences to be used can be designed appropriately for any given organism with which the nucleic sequence data is known or can be determined accurately to perform the test in the present invention. In order to have a detectable signal for the hybridization, a concentration of the target nucleic acid should be above 0.5 f moles for avidin-AP conjugate enzyme link color development (other labeling development system may be more sensitive) . Hence if the concentration of the target molecule is low, amplification must be performed before the hybridization process. For demonstration on the validity of the present invention, the PCR amplifications were used for examples given here. Other amplification methods, such as those mentioned elsewhere in this text can also generate similar concentration and therefore should be feasible . In fact, isothermal amplification methods are preferable because the relatively expensive PCR equipment (Thermal Cycler) will not be needed, and the amplification process is much faster. A two-step nested RT-PCR protocol was used for initial development of the SARS diagnosis kit. The reversed transcription was done with the first primer pair: 5' -ATGAATTACCAAGTCAATGGTTAC-3' and 5' -CATAACCAGTCGGTACAGCTAC-3' with positive control RNA of about 2xl0^"5 copies using the Thermoscript RT-PCR system and Platinum Taq DNA polymerase, followed by the first round of PCR. The 2^nd round PCR was done using lul of the 1:10 dilution of the first round product in 50ul reaction volume. The thermal cycling program used for both round was the same : (1)95°C, 5 min; (2) 95°C, 10 sec;(3)56°C, lOsec; (4) 72°C, 30sec (5)39 cycle repeats on steps 2 to 4, followed by 72°C, 10 min final extension time. A unique two-stage PCR protocol (see Table I) was developed to amplify to the lowest detection limit of a single target molecule and reproducibly for 2 molecules with consistency. The nested PCR was done in one tube for the first and the second round PCR. This protocol not only simplifies the sample handling, it also saves time, and most importantly, by removing the second round sample preparation, the possibility of cross contamination which is the main concern for all PCR processes is reduced. Figure 3 shows the typical amplification results evaluated by agarose gel electrophoresis for such amplifications. The process was a two-stage single program nested PCRamplification in which the outside primer pair was designed to have a higher annealing temperature for the initial amplification of 10 to 20 cycles followed by a lower annealing temperature amplification cycles . As shown in Figure 3, this process has produced a much more specific (less non-specific band(s) was obtained other than the target sequence) result even at the limit of its detection.

Figure 4 showed the hybridization result for SARS. Evidently, the hybridization result was very specific and there was no cross (non-specific) reaction for the negative control. Double-blind studies were performed on a panel of 20 clinical samples, i.e. diluted samples of the archive samples that had been detected by the Real Time PCR. The results as shown in Table II indicated that 18 of the 20 samples were correct. The two positive samples that were not successfully amplified were in fact below the claimed detection limit of one copy each. Despite this result, the sensitivity and specificity achieved by the two-stage PCR amplification and flow-through hybridization was 90% and 100% respectively. Table I . Two-stage single program nested PCR protocol

PTC-200 Thermal cycling profile

Table II. Analytical Detection of SARS Comparing the HybriMax Test to Real-time PCR

** The concentration of one of these two samples was only 1 copy per microliter (i.e. a maximum of 1 copy per reaction)

One-step and nested PCR were also tested (the nested PCR cycling program is given in Table III below) . Based on the known sequence data of the SARS variants published in the GeneBank, a set of ASO (or SNP) oligo-probes and PCRprimers, which are listed in the Appendix, were designed. In principle, ASO or SNPs probe (s) and amplification primers of any organisms with adequate sequence data can be designed to perform such genetic analysis, and can be detected by the flow-through hybridization method. The results of hybridization were obtained within minutes. These SNP oligo-probes were used as the low-density array for detecting multiple SARS variants on a single membrane for the SARS causing coronus viral nucleic acid diaqnosis.

Figure 5 showed the array of ASO probes dotted onto the membrane for hybridization detection with target samples. Using the three archived known variant samples as a control panel, the protocol of the fast flow-through hybridization was developed successfully. The typical result is shown in Figure 6. To evaluate the accuracy of this test, 10 random clinical samples were tested and the results were confirmed by DNA sequencing. The results shown in Table IV below indicated 100% agreement. Table III. Nested PCR Amplification Cycling Program Specifically for SARS Corona Virus

Table IV. Analytical Detection of SARS Variants Comparing the HybriMax Test to DNA Sequencing

(II) Diagnosis of the Upper Respiratory Track infectious viruses , including SARS coronus virus

Detailed sequences of the various viruses isolated as the causative pathogens for the upper respiratory track infection have been published and the sequence data submitted to GeneBank . The ASO probes of all the 11 species listed in this invention were designed for the production of the test array (shown in Figure 7) . PCR primers for the amplification of the pathogens and the probe sequences are listed in the Appendix. The multiplex RT-PCR amplification was developed using the cycling program given in Table V and the analytical sensitivities for the detection limit of each of the viruses have been evaluated using serial dilution of the in vitro transcribed products from the respective viral clones. The test specificity was evaluated with the use of nucleic acid samples and probes of 10 other respiratory tract pathogens (respiratory syncytial virus (RSV) , parainfluenza virus type 1, 3 & 3b (PIV 1, 3 & 3b) , Mycoplasma pneumoniae, Chlamydia pneumoniae, enterovirus, influenza A & B, adenovirus, metapneumovirus). The flow through hybridization protocols are similar to that described above for the SARS diagnosis . Figure 8 showed the typical results of the test array with 4 of the 11 the viruses for such evaluation. This figure shows examples of the specific HybriMax-based hybridization of SARS coronavirus and other pathogens PCR products to SARS coronavirus probe and other specific probes respectively. Membrane positions of probes are as follows: 1 Respiratory syncytial virus (RSV); 2 Enterovirus (EN); 3 Adenovirus (Adeno) ; 4 Parainfluenza virus type 1 (PIV1); 5 Parainfluenza virus type 3 (PIV3); 6 Parainfluenza virus type 3b (PIV3b) ;7 InfluenzaA (Inf A) ; 8 Influenza B (Inf B) ; 9 SARS coronavirus (SARS);10 Metapneumovirus (Meta); 11 Mycoplasma pneumoniae (MP) ; 12 Chlamydia pneumoniae (CP) .

Table V. Multiplex One-step RT-PCR Amplification Program for the Upper Respiratory Track Viral Detection

Total volume 25 μl *6-plex primer mix contains primers for SARS coronavirus (SARS1 & SARS2), influenza A & B viruses, parainfluenza virus type 1 & 3, respiratory syncytial virus.

Evidently the process did not produce non-specific cross reaction. To ensure the validity, this array test format was evaluated with archived clinical samples provided by hospital. In evaluating the HybriMax system, immunofluorescence, rapid antigen test or viral culture was used as the reference method to determine the relative false-positive and false-negative rates. Of the 57 samples, the proportion of immunofluorescence/rapid antigen test/viral culture negative results that were positive by HybriMax was 1/5, resulting in a relative false-positive rate of 20% and a specificity of 80%. Conversely, the proportion of immunofluorescence/rapid antigen test/viral culture positive results that were negative by HybriMax was 2/52, resulting in a relative false-negative rate of 3.8% and a sensitivity of 96.2%. The actual test data and the summary are given in Table VI & VII below. The false negative results from Samples 225433 and 225647 were due to PCR failure (not due to hybridization failure) which may have been caused by the degradation of the samples during storage.

The ability to simultaneously and rapidly detect the other viruses causing the SARS like syndrome is of utmost important because this will be the most effective way for triage patients for effective treatment and prevent unnecessary spread of the serious communicable infectious SARS disease. Other than identify the general species as list in Figure 7, the present invention can also be extended to include simultaneously identify variants of these viruses in one single membrane test strip to enable physicians to prescribe the most appropriate therapy to save lives . The rapid and simplicity of test which can be carried out onsite is unique to the present invention.

Table VI . Clinical evaluation of Multiple Pathogen Multiplex PCR and Hybridization System Targeting 57 Patient Samples to Determine Sensitivity and Specificity

Fifty seven archived nasopharyngeal aspirates (NPA) samples stored at -20°C for over 3 months were tested. The samples were also tested with Binax FDA cleared fast test kits for Influenza A, Influenza B and RSV and immunofluorescence assay for Adenovirus, PIV 1 and PIV 3.

Table VII. Analytical Detection of SARS Coronavirus, Influenza A Virus, Influenza B Virus, PIV 1, PIV 3, RSV Comparing the HybriMax Test to Immunofluorescence/Rapid Antigen Test/Viral Culture

Sensitivity = No of True Positive ÷ (No of True Positive + No of False Negative) = 96.2%

Specificity = No of True Negative ÷ (No of True Negative + No of False Positive) = 80% Materials : Probe : 1. Amino modified oligonucleotide capture probes - BNIcapture (stock at (given in appendix) , -20°C, 1 nmol/μl, dilute to 10 μM with 0.5 M sodium bicarbonate)

Membrane Arrays preparation reagents :

1. Biodyne C membrane (Pa ll) (used for probe immobiliza tion to capt ure the target labeled DNA samples used a s the ma trix for the low-densi ty array)

2 . Preactivation solution: 0.1 M HC1

3. 20% EDAC (l-ethyl-3- (3-dimethylaminopropyl) carbodimmide hydrochloride) (store at 4°C) (Just before use as a 20% solution in water) (Sigma 89H0804)

5. Oligonucleotide application buffer: 0.5 M sodium bicarbonate, pH 8.4

6. Membrane quenching solution: 0.1 M NaOH

7. Hybridization and wash buffer: 2X SSC/0.1%SDS

Color Development reagents :

1. StabilGuard^® - (SurModics SGOl-1000) (stored at 4°C)

2. StabilZymeAP^® - (SurModics SAOl-1000) ( 4°C)

3. TBST - 50 mM Tris, 150 mM NaCl, 0.1% Tween20 (v/w) , pH 7.5 (4°C) 4. Avidin-Alkaline Phosphate conjugate (from hen egg white) , 1 mg/ml (4°C) 5. NBT/BCIP ready-to-use tablets (Roche 1697471) ( 4°C) Others

1. 15 ml centrifuge tube

2. 1 ml, 200 μl, 0.5 μl pipette tips 3. pipettes (P1000, P200, P100, P2)

Instruments :

1. DNA Flow-Through Hybridization Device (DNA HybriMax)

2. Heat block or water baths 3. Thermalcycler for PCR

Methods :

Memebrane Array preparation (Covalent attachment of amino modified probes to Biodyne C membrane filter) 1. Briefly rinse the membrane with 0.1 M HCl, blot, then soak in freshly prepared 20% EDAC (lg EDAC in 5 ml Milli-Q dH₂0) for 15 min .

2. Rinse the filters in deionized water and blot to remove excess water. The carboxyl groups on the membranes are activated at this point and the filters must be processed immediately (within 0.5 hr)

3. Place filters on Parafilm, apply 0.5 μl 10 μM (5μM/dot) of modified probes on the membrane.

4. Incubate the filters for 15 min at room temperature. 5. Quench the covalent attachment reaction, rinse the membranes thoroughly for 10 min with 0.1 M NaOH 6. Wash the membrane with four to five rinses in deionized water. 7. The filters may be used immediately or air-dried for subsequent use. When dry, the membrane appears to be stable for indefinite periods. (Store the membrane at dark)

PCR Product- PCR amplification of the target nucleic aσid(s)

1. SARS and other upper respiratory track infectious viral PCR products in the examples shown in this invention were labeled with biotin-labeled primers.

2. The PCR amplification was done using either the MJ p200 or the ABI 9700 thermalcycler (in principle, any other thermal cyclers can be used for the amplification with appropriate optimization of the cycling protocols) . Single or nested PCR have been tried successfully with the one-step or the two-step processes.

Procedures for Hybridization of PCR products to filters

(Note: Cover the unused well with parafilm or alumnium foil)

1. Denature the DNA by heating at 95°C for 5 min.

2. Chill on ice for at least 2 min.

3. Place the membrane on HybriMax and set the temperature to 36°C. 4. Prehybridize the membrane with 1ml 2X SSC/0.1%SDS.

5. Add 10 μl of PCR product to 0.5 ml 2x SSC/0.1%SDS in 1.5 ml eppendorf tube . 6. Add 0.5 ml of the denatured DNAs into designated well. Incubate for 5 min for better hybridization. 7. Pump the solution out (PUMP ON).

8. (Keep PUMP ON) Wash the membrane with 2.4 ml 2X SSC/0.1%SDS.

9. PUMP OFF. Color Development procedure

I. Add 0.5ml of StabilGuard^® to the membrane. Pump off the solution completely . 2. Pump off.

3. Block the membrane with 0.5 ml StabilGuard⁰ for 5 mm. Pump out the solution

4. Dilute avidm-AP conjugate by adding 1 μl AP stock (1 mg/ml) to 3 ml StabilZyme AP. (1:3000 dilution) 5. Add 0.5 ml diluted AP-conjugate to each well. Incubate for 3 mm for better reaction.

6. Pump out the solution (PUMP ON) .

7. (Keep PUMP ON) Wash the membrane with 3.2 ml TBST (NOTE: Other developing reagents such as Avidin-colloidal gold or magnetic particles or fluorescence labels or other can be used for direct detection and save the following steps . Using these direct detection systems, the time will be less then 1 minute.)

8. PUMP OFF. Dissolve 1 NBT/BCIP ready-to-use tablet in 10ml double distilled water. 9. Add the dissolved NBT/BCIP to each well (2.5 ml for each well) .

10. Incubate until the color develops. (Around 5 minutes)

II. PUMP ON. Wash the membrane with 3ml 2XSSC/0.1%SDS followed by lml dH₂0 after the color completely developed.

As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof. Accordingly, the scope of the invention is to be construed in accordance with the substance defined by the following claims.

References

1. DrostenC. etal., identificationofa novel coronavirus in patients with severe acute respiratory ayndrome . N. Engl . J. med., 348: 1967-1976.

2. Guan Y, etal., Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 2003; 302: 276-8.

3. Poon LLM et al., Early diagnosis of SARS coronavirus infection by real time RT-PCR. J Clin. Virol 2003; 28: 233-8.

4. Grant PR et al., Detection of SARS coronavirus in plasma by real time RT-PCR. N Eng J Med 2003; 349: 2468-9.

5. Wong CW, Albert TJ, Vega VB, Norton JE, Cutler DJ, Richmond TA, Stanton LW, Liu ET, Miller LD, Tracking the evolution of the SARS coronavirus using high-throughput, high-density resequencing arrays. Genome Res. 2004; 14 (3) : 398-405. 6. MarraMA, Jones SJ, AstellCR, HoltRA, Brooks-Wilson A, Butterfield YS, Khattra J, Asano JK, Barber SA, Chan SY, Cloutier A, Coughlin SM, Freeman D, Girn N, Griffith OL, Leach SR, Mayo M, McDonald H, Montgomery SB, Pandoh PK, Petrescu AS, Robertson AG, Schein JE, Siddiqui A, Smailus DE, Stott JM, Yang GS, Plummer F, Andonov A, Artsob H, Bastien N, Bernard K, Booth TF, Bowness D, Czub M, Drebot M, Fernando L, Flick R, Garbutt M, Gray M, Grolla A, Jones S, Feldmann H, Meyers A, Kabani A, Li Y, Normand S, Stroher U, Tipples GA, Tyler S, Vogrig R, Ward D, Watson B, BrunhamRC, Krajden M, PetricM, Skowronski DM, Upton C, Roper RL. The Genome sequence of the SARS-associated coronavirus. Science. 2003 May 30; 300(5624) :1399-404. Epub 2003 May 01.

7. Notomi T et al.' Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000; 28: E63.

8. Hong Kong DNA Ltd (written by Joseph W. 0. Tam) , DNA Flow-Through Hybridization Device User Manual

9. Methods in Molecular Biology Vol. 28: Protocols for Nucleic Acid Analysis by Nonradioactive Probes, Chapter 33: Analysis of Gene Sequences by Hybridization of PCR - Amplified DNA to Covalently Bound Oligonucleotide Probe. The Reverse Dot Blot Method by Ernest S. Kawasaki & Farid F. Chehab p. 225 - 236).

10. Chan V. T.-W. , Fleming K. A. and McGee J. O'D. (1985). Detection of subpicogram quantities of specific DNA sequences on blot hybridization with iotinylated probes . Nucleic Acids Research, 13: 8083-8091.)

U.S. Patent Documents : 1. USPTO # 5,741, 647 Tam, Joseph Wing On

2. USPTO # 6, 020, 187 Tam, Joseph Wing On

3. USPTO # 6,727, 063 Lander, et al. Appendix (A) SPEICES LISTING for testing kit used in the present invention:

(i) Coronavirus (SARS) Variants used in the invention: Known genoptypes : Urbani ; HKU-39849; CUHK-sulO; SIN2500; SIN2677; SIN2679; SIN2748; SIN2774; T 1; ZJ01; TOR2 ; CUHK- 1 ; BJ01 Other variants: (once the sequences of any new type is known, the present invention can be used to develop the necessary protocols for the detection of its presence)

(ii) Multiple viral test panel (membrane array) used in this invention" 1. Respiratory syncytial virus (RSV) 2. Enterovirus (Entero) 3. Adenovirus (Adeno) 4. Parainfluenza virus type 1 (PIV1) 5. Parainfluenza virus type 3 (PIV 3) 6. Influenza A (Inf A) 7. Influenza B (Inf B) 8. SARS coronavirus (SARS) 9. Metapneumovirus (Meta) 10. Mycoplasma pneumoniae (M. Pneu) 11. Chlamydia pneumoniae (CP)

(iii) Other viral species: Human coronavirus 229E Animal Viruses : Canine coronavirus Feline infectious peritonitis virus Porcine transmissible gastroenteritis virus Porcine epidemic diarrhea virus Bovine coronavirus Porcine hemagglutinating encephalomyelitis virus Rat sialodacryoadenitis virus Mouse hepatitis virus Turkey coronavirus Avian infectious bronchitis virus

(B) Nucleic Acid Sequences

(i) Capture probe sequences:

1. SARS - GGGTACTAACCTACCT 1. CuBJ_laCap - CGTTTGACAACCCTG

2. CuBJ_laWTCap - TTTGGCAACCCTGT

3. CuBJ_lbCap - TCAAGACACTTGGGG

4. CuBJ_lbWTCap - TCAAGACATTTGGGG 5. Tor2Cap - ACCTTGCGCTTTTG

6. Tor2WTCAP - ACCTTGCTCTTTTGG 7. RSV - CCTGCATTAACACTAAATTC

8. PIV1 - TACCTTCATTATCAATTGGTAAGTCAATATATG

9. PIV3 - AAAATTCCAAAAGAGACCGGC 10. PIV3B - ACAGAACACCARAACAACAA

11. M. pneumoniae - ACTCCTACGGGAGGCAGCAGTA

12. C. pneumoniae - TCTTGCTACCTTCTGTACTAA

13. Enterovirus - GAAACACGGACACCCAAAGTA

14. Influenza A - GTCCTCATCGGAGGACTTGAATGGAATGAT 15. Influenza B - GTCAAGAGCACCGATTATCAC

16. Adenovirus - CTCGATGACGCCGCGGTGC

17. Metapneumovirus - GAAGTTTGTTCATTGAGTATGG

(ii) PCR Primer sequences 1. SARS1 - ATGAATTACCAAGTCAATGGTTAC

2. SARS2 - CATAACCAGTCGGTACAGCTAC

3. SARS3 - GAAGCTATTCGTCACGTTCG

4. SARS4 - CTGTAGAAAATCCTAGCTGGAG

5. CuBJ_laF - TCAGGATTTATTTCTTCCATTTT 6. CuBJ_laR - GCAGCAAAATAAATACCATCC

7. CuBJ_lbF - GAGCCATTCTTACAGCCTTT

8. CuBJ_lbR - TACCATTTTCATCATACTTGAGC 9. SIN2696F - CAACCATTTCAACAATTTGG 10.SIN2696R - AGCATTTGTTCCAGGTCTAAT

11. RSV forward - TTAACCAGCAAAGTGTTAGA

12. RSV reverse - TGTTATAGGCATATCATTGA 13. PIV1 forward - ATTTCTGGAGATGTCCCGTAGGAGAAC 14.PIV1 reverse - CACATCCTTGAGTGATTAAGTTTGATGA 15. PIV3 forward - AGAGGTCAATACCAACAACTA

16. PIV3 reverse - TAGCAGTATTGAAGTTGGCA

17. M. Pneumoniae forward - AAGGACCTGCAAGGGTTCGT 18. M. pneumoniae reverse - CTCTAGCCATTACCTGCTAA

19. C. pneumoniae forward - TGACAACTGTAGAAATACAGC 20. C. pneumoniae reverse - CGCCTCTCTCCTATAAAT 21. Enterovirus forward - TCCTCCGGCCCCTGAATGCG 22. Enterovirus reverse - ATTGTCACCATAAGCAGCCA 23. Influenza A forward - AAGGGCTTTCACCGAAGAGG

24. Influenza A reverse - CCCATTCTCATTACTGCTTC

25. Influenza B forward - ATGGCCATCGGATCCTCAAC

26. Influenza B reverse - TGTCAGCTATTATGGAGCTG

27. Adenovirus forward - ATGACTTTTGAGGTGGATCCCATGGA 28. Adenovirus reverse - GCCGAGAAGGGCGTGCGCAGGTA

29. Metapneumovirus forward - CATGCCCACTATAAAAGGTCAG

30. Metapneumovirus reverse - CACCCCAGTCTTTCTTGAAA

Claims

What is claimed is:

1. A method for the detection of a pathogen, comprising the steps of: a. selecting at least one appropriate target sequence from the pathogen; b. screening said sequence to obtain at least one set of appropriate primer (s) for amplification; c. selecting an appropriate probe from the sequence; d. immobilizing the allelic-specific-oligonucleotide site of each sequence onto a solidmatrix suitable for capturing the target sequence; e. amplifying the sequence (s) wherein the steps (d) and (e) are performed separately; f . performing blot analyses on each sequence; and g. comparing the results of the blot analysis with using positive or negative controls.

2. The method of claim 1, wherein the sequence is a gene, pathogen or allelic sequence.

3. The method of claim 1, wherein the pathogen is one of the upper respiratory viruses.

4. The method of claim 1, wherein the primer and the sequence-specific-oligonucleotide site are obtained by screening data from the GenBank or by sequencing the target sequence or in combination thereof.

5. The method of claim 1, wherein the target sequence comprises a gene, nucleic acid or DNA sequence.

6. The method of claim 1, wherein the probe is a single oligo-nucleotide .

7. The method of claim 1, wherein more than one probe is selected.

8. The method of claim 1, wherein the probe is a single nucleotide polymorphism probe.

9. The method of claim 1, wherein the solid matrix is a membrane.

10. The method of claim 1, further comprising amplifying the target sequence having a concentration less than 0.5 f moles.

11. The method of claim 1, wherein the amplification step can be performed by polymerase chain reaction amplification, isothermal amplification, nested real-time polymerase chain reaction amplification, two-step nested real-time polymerase chain reaction amplification.

12. The method of claim 11, wherein the amplification step comprises: a. selecting an appropriate primer pair; b. performing reverse transcription on the primer pair; and c. performing the first and second round of PCR in a single tube .

13. The method of claim 12, wherein the second round of PCR is performed with a thermal cycling program having the following thermal cycling profile: (1) 95°C, 5 min; (2) 95°C, 10 sec; (3) 56°C, lOsec; (4) 72°C, 30sec; (5) 39 cycle repeats on steps 2 to 4, followed by 72°C, 10 min final extension time.

14. The method of claim 12, wherein the second round PCR was done using lul of the 1:10 dilution of the first round product in 50ul reaction volume.

15. The method of claim 12, wherein the reverse transcription is performed with positive control RNA of about 2xl0^~5 copies using the Thermoscript RT-PCR system and Platinum Taq DNA polymerase .

16. The method of claim 12, wherein the second round of PCR is performed with a thermal cycling program having the following thermal cycling profile: (1) 95°C - 3min; (2) 95°C , lOsec; (3) 60°C, lOsec (drop by l°C/cycle) ; (4) 72°C, 30 sec; (5) 9 cycle repeats on steps 2 to 4; (6) 95°C, lOsec; (7) 56°C, lOsec; (8) 72°C, 30sec; (9) 9 cycle repeats on steps 6 to 8; and (10) Store at 4°C.

17. The method of claim 12, wherein more than one target sequence from at least one pathogen is amplified.

18. The method of any one of the above claims, wherein the pathogen is an upper respiratory virus.

19. The method of claim 18, wherein the virus consists of the group selected from respiratory syncytial virus (RSV), parainfluenza virus type 1 (PIVl), parainfluenza virus type 3 (PIV3), parainfluenza virus type 3B ( PIV3B) , Mycoplasma pneumoniae (MP) , Chlamydia pneumoniae (CP) , Enterovirus (EN), Influenza A (Inf A) , Influenza B (Inf B) ; Adenovirus (Adeno) , Metapneumovirus (Meta), and SARS coronavirus (SARS).

20. The method of claim 1, wherein more than one pathogen is used.

21. The method of claim 1, wherein a pair of primers are used.

22. The method of claim 1, completed within two hours.

23. The method of claim 1, complete within minutes.

24. The method of claim 1, completed within 20 minutes using enzyme link color development.

25. The method of claim 1, completed within between 2 to 3 minutes with using use colloidal gold or florescence dye.

26. The method of claim 1, used for identification of pathogens.

27. The method of claim 26, wherein the pathogens are SARS causative pathogens .

28. The primer, not previously known, used in the method of any of claims 1-27.

29. The probe, not previously known, used in the method of any of claims 1-27.

30. A kit for detection of pathogen comprising a compartment containing the primer of claim 28 or the probe of claim 29, or a combination thereof.

31. A machine or computer capable of carrying out the method of any of claims 1-27.

32. A software dictating the complete or part of the method of any of claims 1-27.

33. The method of claim 1, wherein the solid matrix is a low-density array for detecting multiple SARS variant.

34. The method of claim 1, wherein the solid matrix is a viral detection array for detecting upper respiratory tract infection.

35. The method of any one of claims 33 or 34, wherein the array is on a single membrane.

36. An array, not previously made, used in any of claims 33-35.

37. The method of claim 1, wherein steps (d) and (e) are performed sequentially or concurrently.