CA2231271A1 - Molecular diagnosis of viral infections - Google Patents
Molecular diagnosis of viral infections Download PDFInfo
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Abstract
Reverse transcription employing pooled short oligonucleotides of random sequence as primers is used to obtain cDNA from viral RNA in a extract of cells suspected of being infected by RNA pathogens. The cDNA may be amplified in a single PCR procedure employing primers specific for two or more pathogens in order to detect the presence of multiple RNA pathogen infection in the cells.
DNA so amplified may incorporate bromo-deoxyuridine which facilitates a convenient and non-radioactive means for detecting the amplified product.
DNA so amplified may incorporate bromo-deoxyuridine which facilitates a convenient and non-radioactive means for detecting the amplified product.
Description
CA 0223l27l l998-03-0~
MOLECULAR DIAGNOSIS OF VIRAL INFECTIONS
This invention relates to the detection of the genetic material of RNA pathogens and in particular, multiple RNA
respiratory pathogens.
Laboratory testing to identify disease causing pathogens in clinical specimens is necessary in a variety of circumstances. Test results may be useful for guiding physicians in terms of appropriate patient management and medication, gathering information necessary for epidemiology and public health purposes, and research into mechanisms of infections caused by microorganisms.
Conventional laboratory methods of viral diagnosis such as culturing the pathogen, electron microscopy, immunodetection of the pathogen and serology are subject to significant limitations. Such limitations may be brought about by factors such as poor replication of pathogens in culture, requirement for specialized and expensive equipment (e.g. electron microscopy) and, existence of numerous serotypes of a particular pathogen.
Amplification of genetic material derived from a respiratory pathogen provides a sensitive and specific meanE3 for detecting such pathogens. Reverse transcription (RT) of viral RNA followed by DNA amplification in the CA 02231271 1998-03-0~
polyrnerase chain reaction (PCR) has been shown to be a sens:Ltive diagnostic procedure for detection of several RNA
virus respiratory pathogens: Gilbert, L.L., et al. (1996) Diagnosis of Viral Respiratory Tract Infections in Children by using a Reverse Transcription-PCR Panel; Journal of Clin:Lcal Microbiology 34:140-143. However, such procedures are complex and requi:re a significant length of time to be carr:Led out. This is disadvantageous in a commercial clin:Lcal laboratory setting, particularly in situations where the diagnostic procedure must detect multiple RNA
pathogens. In addition to upper and lower respiratory tracl infections (e.g. common cold, pneumonia), multiple RNA respiratory pathogens may infect a single patient show:Lng symptoms of asthma or exercise-induced bronchoconstricton (EIB).
Summary of Invention This invention provides a method to obtain RNA
pathogen cDNA through reverse transcription (RT) wherein an extract of cells suspected of containing a RNA pathogen is used in a RT reaction in which pooled short oligonucleotides of random sequence are used as a source of RT ~rimer. The RNA pathogen may be a RNA respiratory pathogen.
While the method of this invention may be used to obta:in CDNA from the RNA of any pathogen, the method is CA 02231271 1998-03-0~
particularly suited to the diagnosis of multiple RNA
pathogen infections since the RT reaction does not depend upon use of specific oligonucleotide primers. Rather, the use of random oligomers as primers permits reverse transcription of RNA from multiple sources present in a single sample.
Use of random oligomers as RT primers is known for the dete,-tion of RNA from single RNA pathogen sources; see:
M.H. Vodkin et al. (1994) "PCR - Based Detection of Arboviral RNA from Mosquitoes Homogenized in Detergent", BioTechniques 17:114-116; R.F. Meyer et al. (1991) "Rapid and Sensitive Detection of Foot and Mouth Disease Virus in Tissues by Enzymatic RNA Amplification of the Polymerase Gene", Journal of Virological Methods 34:161-172; and, D.K.
Howe et al . ( 1992) "Use of the Polymerase Chain Reaction for t:he Sensitive Detection of St. Louis Encephalitis Viral RNA", Journal of Virological Methods 36:101-110. Also, use of random oligomers as RT primers has been demonstrated for the l~etection of multiple RNA pathogens from non-cellular sour~es; see: Yu-Li Tsai, et al. (1994) "Detection of Polio Virus, Hepatitis A Virus, and Rotavirus from Sewage and Ocean Water by Triplex Reverse Transcriptase PCR", Applied Envi:ronmental Microbiology 60:2400-2407.
This invention provides a method to obtain RNA
resp:iratory pathogen cDNA through reverse transcription CA 02231271 1998-03-0~
(RT) of genetic material obtained from a respiratory source comprising the steps of:
(a) extracting RNA from cells from a respiratory source; and (b) using RNA from step (a) as a template in a RT
reaction in which pooled short oligonucleotides of random sequence are used as a source of RT primer.
RNA respiratory pathogens detectable by the method of this invention include single stranded RNA viruses of which the following are examples: picoronavirus (rhinoviruses and/or enteroviruses); parainfluenza viruses (PIV);
respiratory syncytial virus (RSV); influenza viruses A, B
and C; and, coronaviruses OC43 and 229E.
Cells from a respiratory source may be cells of any respiratory specimen including nasopharyngeal or tracheal washes and aspirates, bronchoalveolar levage fluid, and lung tissue specimens.
Methods for extracting RNA from cells and cell suspensions are well known in the art, such as the procedures described in: Chomczynski, P. and N. Sacchi (1987) Single-Step Method of RNA Isolation by Acid Guan:idinium Thiocyanate-Phenol-Chloroform Extraction; Anal.
Biochem. 162:156-159. A preferred method of RNA extraction by cell lysis is described below.
CA 02231271 1998-03-0~
Methods for performing RT are well known in the art and commercial RT kits are readily available. However, rather than using viral specific oligonucleotide primers, this method employs a pool of random oligomers as a source of the RT primers. The pool of random oligomers should be assernbled such that as many of the possible permutations of random sequences are provided in the pool. This is most conveniently done by mixing different oligomers having the short:est possible sequence that will function efficiently as a RT primer. Hexamers will efficiently act as RT
primers at normal temperatures (e.g. 37~C) and there are only 4,096 possible permutations. Pools of random hexamers for use as primers are commercially available (e.g Pharmacia, Montreal, Canada).
This invention also provides a method of amplifying DNA derived from two or more sources by PCR in a single samp~Le. For example, the DNA may be cDNA derived from two or more RNA respiratory pathogens. According to this aspect of the invention, DNA derived from different sources is annplified in a single sample using primers specific for two or more pathogens. Preferably, the primers are selected such that the amplified DNA associated with different pathogens will be of such differing lengths as to be d:iscernible following usual separation procedures such as electrophoresis. PCR amplification procedures and reagents are well known in the art. Combination of the above described method of RT using random oligomers with CA 02231271 1998-03-0~
multiple PCR amplification in a single sample provides a great:ly streamlined yet efficient process for obtaining detectable levels of genetic material derived from multiple RNA pathogens.
This invention also provides an efficient method for amplification of DNA, such as cDNA derived from RNA
respi.ratory pathogens r whereby the resulting amplified DNA
may be detected by an immunostaining procedure which does not require the use of radio-labelled probes yet is comparable in sensitivity to the latter procedure. This method facilitates the above described method of multiple PCR amplification in cL single sample since its sensitivity permits the detection of product which is present in a minut:e amount as compared to other amplified product in the samp]e. The method was described by the inventors in: A
Nonradioactive Method for Rapid and Sensitive Detection of Polymerase Chain Reaction Products by use of Bromo-Deoxyuridine (1996) Modern Pathology 9:849-853.
This invention also provides kits comprising reagents for carrying out the aforementioned methods. A kit for performing the above described RT method will comprise a pool of random oligomers for use as RT primers, preferably a pool of random hexamers. When the kit is to be used for carrying out the above described method of multiple PCR
amplification, it will comprise RNA respiratory pathogen speciLfic primers. Such a kit will preferably also comprise CA 02231271 1998-03-0~
a dNTP mixture in which bromo-deoxyuridine (Br-dUTP) is subst:ituted for dTTP. Such a kit would also preferably include an anti-Br-dU antibody and the remainder of a ELISA
system whereby amplified DNA containing Br-dU may be readily detected (e.g. by enzyme-chemiluminescence).
Detailed Description of the Invention Detailed descriptions of embodiments of this invention are cet out in the accompanying examples. Unless otherwise stated, autoclaved plasticware (at least 20 min. on dry heat, 30 psi, 132~C) is used, and aqueous solutions contain 0.1~ diethylpyrocarbonate (DEPC) as an RNAse inhibitor. To prevent inadvertent exogenous RNAse activity from handling of specimens, investigators should wear latex gloves that are changed several times during the course of the procedure.
RNA ~xtraction. Total RNA is extracted from a thawed cell suspension (e.g., nasopharyngeal swabs, bronchoalveolar lavage fluid) by use of the RNeasyTM spin column, lysis and wash buffers (Qiagen Corporation) as follows: a 300 ~L
aliquot of the specimen is added to an autoclaved 1.5 mL
Eppendorf tube, followed by addition of 300 ~L of lysis buffer RLT, 600 uL of 70~ ethanol and mixing by agitation on a vortex. This 1.2 mL sample is applied onto the RNeasyTM spin column and centrifuged for 15 s at 10,000 rpm.
The flowthrough is discarded. Seven hundred ~L of wash CA 02231271 1998-03-0~
buffer RW1 is added to the spin column, the sample is centrifuged as above and the flowthrough is discarded. The spin column is placed in a new 2 mL collection tube. Five hundred ~L of wash buffer RPE is pipeted onto the spin colun~m, and the sample is centrifuged as above, with the flowthrough being discarded. Wash Buffer RPE (500 ~L) is pipet.ed onto the spin column and the sample is centrifuged at full speed for 2 min. The spin column is transferred to a new collection tube and the RNA is eluted by addition of 30 ~l of DEPC-distilled, deionized water and centrifugation at 10,000 rpm for 1 min.
In cases where RNA is extracted from tissue specimens (such as frozen lung tissue) the commercially available TRIZC)LTM reagent may be used (see United States patent No. 'i,346,994.
Reverse transcription (RT). To remove secondary structure from RNA, a 10 ~L aliquot from the RNA sample as prepared above is heated to 68"C for 10 min., then quick-chilled on ice. This linearized RNA is added to an Eppendorf tube that contains 10 ~L of the following aqueous RT reaction solut:ion at room temperature: 10 mM Tris-HCl (pH 8.3);
50 mM KCl, 5 mM MgCl2, 10 mM pooled deoxynucleotide triphosphates (dNTPs),(Pharmacia) 1 ~L of 50 ~M random hexamers to act as "universal" primers for RT, 200 units Moloney murine leukemia virus reverse transcriptase, (Pharmacia) and 20 units of RNAse inhibitor (Pharmacia).
CA 02231271 1998-03-0~
g RT occurs in the closed Eppendorf tube at 37~C for 60 min., after which time the tube is heated at 95~C for 5 min. to inactivate the reverse transcriptase. The specimen is then placed in ice until used for PCR.
5 PCR almplification. PCR amplification is carried out in a 50 IlL, reaction volume containing: all or part of the 20 ~L
aliquot from the RT procedure; 10 mM Tris-HCl, pH 8.3;
50 mM KCl; 5 mM MgCl2; 0.2 mM dNTPs, in which an equimolar concentration of 5-bromo-2-deoxyuridine triphosphate 10 (Br-clUTP; Sigma) is substituted for deoxythymidine triphosphate; 2 ~M of oligonucleotide primers specific to two pathogens and, 2 units of Taq DNA polymerase (Gibco-BRL). Specimens undergo brief centrifugation and are overlayed with mineral oil. PCR cycling conditions on 15 a ROBOCYCLER 40TM (Stratagene) involve 35 cycles, each one consisting of denaturation at 94~C for 1 min., annealing at 55~C for 1 min., and extension at 72~C for 1 min. (10 min.
during the last cycle). Samples undergo electrophoresis on l-l.Ci% agarose gels that contain 0.5 ~g/mL ethidium 20 bromide. This is followed by conventional Southern blott:ing on nylon membranes (e.g. Hybond-NTM membranes, Amersham) and fixation of any PCR products (DNA) by cross-linking under ultraviolet light for 2-5 min.
Immunodetection of PCR products. Nylon membranes are 25 pre-equilibrated in 0.1 M Tris buffered saline (TBS), pH
7.5 :Eor 5 minutes at room temperature and then incubated CA 02231271 1998-03-0~
with blocking buffer (0.1 M TBS, pH 7.5 with 5~ non-fat dry milk powder (Carnation)) at 37~C for 15 min. Mouse anti-bromodeoxyuridine antibody (Boehringer Mannheim) is addecl at a final dilution of 1:1,000 in blocking buffer for a 45 min. incubation at 37~C. After three washes of 5 min.
each in TBS, the nylon membranes are incubated at 37~C for min. with peroxidase-conjugated goat anti-mouse immunoglobulin diluted 1:2,000 in blocking buffer. After three final washes in TBS, enzyme-chemiluminescent (ECL) substrate (Amersham) is applied and the membranes are expo~:ed to x-ray film with regular intensifying screens for a period varying from 15 sec. to 5 min.
Example Children selected from a survey of primary school chilclren in Port Alberni, British Columbia, Canada, were identified as having asthma, EIB or as controls. Age and sex distribution of the study participants is shown in Table 1. A nasal swab specimen was taken from each child from the left inferior nasal turbinate with the swab being rotat:ed once. Each specimen was stored in a cryovial on ice and was subsequently refrigerated at -70~C pending further processing. Positive controls for RT-PCR were prepared from total cellular RNA extracted from virus infected cell cultures grown in M199 minimum essential medium (Gibco BRL) supplemented with 5~ fetal calf serum, L-glutamine and vitamins. All viruses and cell lines were CA 02231271 1998-03-0~
obtained from American Type Culture Collection. HEp-2 cells were infected with Long strain type A RSV; MRC-5 cells were infected with human rhinovirus, type lB; Rhesus monkey kidney (MK2) cells were infected with one of:
picornavirus, PIV type 3, Influenza A Weiss-43, human coronavirus OC43, and human coronavirus 229E. Negative controls included blank reagent mixtures which contained all constituents except a nucleic acid template, or with a nucleic acid template prepared from total cellular RNA extracted from uninfected cell cultures of the appropriate type.
The above described procedures using a RNeasy'M spin column were carried out for RNA extraction. Total RNA was eluted into 30 ~L of 0.1~ DEPC containing distilled water.
Two 10 ~L aliquots of total RNA were incubated for ten minut:es at 70~C to remove secondary structure and were quickly chilled on ice. Reverse transcription accordingto the above described procedures using random hexamers as primers were carried out for each aliquot of linearized RNA.
Five ~L aliquots of cDNA from the RT step were added to separate 45 ~L aliquots of PCR reaction buffer containing 10 mM Tris-HC1 (pH 8.3), 5.0 mM MgC12, 10 mM
pooled dNTPs, 20 U Taq polymerase (GIBCO BRL), and 2 ~M of viru~;-specific oligonucleotide primers as shown in Table 2.
PCR was performed on a Robocycler 40TM (Stratagene, La TABLEI. Age and sex distribution of study pantcipants ASTH~LA E~B CONTROL
(n = 21) (n = 16) (n = 33) Male/Female 12/9 6/10 14/19 Median age i S:D 11.8 + 2.0 11.4 + 2.0 11.4 + 1.8 (years) TABLE2.01igon~s used for viral RT-PCR
P~ K
Virus RegionOligonucleotide Sequence (5' - 3')Product Size (bp) RSV nucleocapsid primer I GCGATGTCTAGGTTAGGAAGAA 410 primer 2 GCTATGTCCTTGGGTAGTAAGCCT
probe TAGCTCCAGAATACAGGCATGACTC
PIV F gene primer I AGAGGTCAATACCAACAACTA 20S
5' nont~nslated primer 2 TAGCAGTATTGAAGTTGGCA
region probe AAAATTCCAAAACAGACCGGC
Picornavirus 'i' nontranslated primer I GCA~-l-l~-l~l-l-l~CCC 380 region of primer 2 CGGACACCCAAAGTAG
rhinovirus IB probe GCATTCAGGGGCCGGAG
In~uenza A matri~ protein primer I CAGAGACTTGAAGAT~l~l-l-lGC 212 primer 2 G~l~-l~lCCATGTTATTTGGATC
probc TCCTGTCACCTCTGACTAAGGGGATTTTG
Coronavirus nucleocapsid primer I GGTACTCCTAAGC~ l~-lCG 370 229E primer 2 TGCACTAGGGTTAATGAAGAGG
probe GACTATCAAACAGCATAGCAGC
Coronavirus nucleocapsid primer l AGGAAGGTCTGCTCCTAATTCC 4so OC43 primer 2 TGCAAAGATGGGGAACTGTGGG
probe GGTCTGGCAAAACTTGGCAAGG
CA 0223l27l l998-03-0 Jolla" CA). For each virus tested by the panel, 35 PCT
cycles were performed, each cycle consisting of 1 min.
denaturation of 94~C, 1 min. annealing at 55~C and 2 min.
extension at 72~C (10 min. during cycle 35). A PCR
experiment consisted of testing 10 patient specimens for one of the six asthma-associated viruses.
After completion of PCR, a 45 ,uL aliquot of each PCR
s amp le un de rwent e lect rop ho re sis o n ethidium-bromide-stained, 1-2~ agarose gels (GIBCO BRL), in TBE buffer that contained 44.5 mM Tris-HC1, 44.5 mM boric acid and 1 mM EDTA, Gels were photographed under ultraviolet light using Polaroid 667 ISO 3000 professional print film, after which RT-PCR products underwent Southern transfer onto nylon membranes, hybridization with the virus-specific,32P-labelled oligonucleotide probes as shown in Table 2, and autoradiography.
CA 0223l27l l998-03-0 Viral RNA was detected in 43/70 (61.4~6) of specimens.
Influenza A viral RNA was detected in higher proportion of specimens from clinically stable asthmatic children and children with EIB, in comparison to asymptomatic controls.
Table 3 summarizes the proportion of subject showing evidence of RNA from each of the viruses examined. Table 4 shows that there were similar proportions of subjects in the three groups who had none, one or multiple viruses detected by the RT-PCR panel. The results reveal a high prevalence picoranviral RNA in the three groups of children. The RT protocol may have yielded more virus related cDNA for PCR amplification since DNA could be synthesized from both the genomic RNA of the virus and from viral mRNA transcripts. The random hexamers acting as primers would permit reverse transcription of both forms of virus specific RNA despite differences in polarity.
Exam~)le 2 cDNA samples resulting from the above described RT
procedures are subjected to duplex PCR amplification using the primers shown in Table 5. In each duplex reaction, a cDNA sample is exposed to primers for two pathogens whereby amplification of cDNA associated with each of the pathogens occurs simultaneously in a single sample. The following scheme is employed in order that the amplification products will differ significantly in size to facilitate elect:rophoretic separation:
CA 02231271 1998-03-0~
(a) picornavirus and parainfluenza virus (PIV);
(b) RSV and influenza A virus;
(c) influenza B and C viruses; and (d) Coronaviruses OC43 and 229E.
Detection of amplification products may be carried out according to the radio labelling procedure of Example 1 using labelled probes as shown in Table 2, or by detection of the incorporation of Br-U in amplified DNA as described above and in Example 3.
Simultaneous detection of DNA respiratory pathogens such as adenovirus and Chlamydia may be carried out.
Suitable PCR primers for the latter organisms are shown in Table 5. The procedure employed may involve either RT of total RNA in a sample followed by amplification of cDNA
derived from both RNA and DNA pathogens. Alternatively, a DNA preparation obtained from the respiratory source may be mixed with a cDNA preparation obtained from the above descr-ibed RT step and the single sample is then subjected to multiple PCR amplification of RNA pathogen cDNA and DNA
pathogen DNA according to the methods described above.
CA 0223l27l l998-03-05 TA~BLE 3. Number (perccntage) of subjects showing positive RT-PCR results for each vi us in the panel.
AST~A EIB CON~OL
(n = 21' (n = 16) (n = 33' RSV ~~ 4' ~ (0) 8 '.4 Pl~ (6) 5 i5, Picornaviru~ (25) ,~4 Tnfll~en7 A ~ (3') ( ) Coronav ~i229E 2 J) (0) _ (6) Coronav,~; OC43 3 ~14) ~ (25) J (15) p = 0.034 in co~palison to asymptom~-i~ controls TABLE 4. Number (percent~ge) of subjects showing viral RNA detected by the RT-PCR panel Number of viruses detected by ASTE~IA EIB CONTROL
Rl -PCR panel (n = 21' (n = 16' (n = 33' vimserl-~c-~d ' 6 3 3 ~' virus ~ ~ct~ 5 3 ~ " ' ' 2 vin~es ~etect~d 5 ~- 5 ~3 0 CA 0223l27l l998-03-05 TABLE 5. PCR Primers Picon~(380~product) 5'CGGACACCCAAAGTAG3' 5'GCACTTCTG m CCCC3' P~a~flu~I~(205~) S'AGAGGTCAATACCAACAACTA3' 5'TAGCAGTATTGAAGTTGGCA3' RSV(410~) 5'GCGATGTCTAGGTTAGGAAGAA3' 5'GCTATGTCCTTGGGTAGTAAGCCT3' ~fluo~aA(212~) S'GCTCTGTCCATGTTATTTGGATC3' S'CAGAGACTTGAAGATGTC~ lGC3' ~flu~aB(36~ ~) S'AGCGTTCCTAGl-l-l-lACTTGCAT3' S'GAAAAATTACACTGTTGGTTCGG3' ~flu~aC(425~) S'GCCAGTAATACCAGCAATCTCG3' S'CCCTAATGTCTTGGAGAAGCCAC3' Coro~n~OC43(450~) 5'TGCAAAGATGG~GAACTGTGGG3' 5'AGGAAGGTCTGCTCCTAATTCC3' C~o~n~229E(370~) 5'TGCACTAGGGTTAATGAAGAGG3' S'GGTACTCCTAAGCCTTCTCG3' Ad~o~n~(300~) S'CAGCACGCCGCGGATGTCAAAGT3' 5'GCCGCAGTGGT~TTACATGCACATC3' Chl~my~a(l45~) 5'GCCTGTAGGGAAT(CG)CAGCT(CG)AA(CT)CA3' S'GTCGAAAACAAAGTC(AT)CC{AG)TAGTA3' CA 0223l27l l998-03-0 Example 3 The above described PCR procedures are carried out using the method described by the inventors at: (1996) Modern Pathology 9: 849-853 in which dTTP is substituted by Br-dUTP in the PCR reaction followed by electrophoresis, Southern Blotting and immunostaining as described above.
Duplex RT-PCR for RSV and influenza A virus in the presence of Br-dUTP was carried out with a constant amount of RSV
and serial 10-fold dilutions of influenza A virus. The results yield a distinct influenza A band (212 bp product) that may be detected in specimens in which 1:1000 of the starting amount of viral RNA was mixed with a large excess of RSV RNA ( 410 bp product).
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 attached claims.
CA 0223l27l l998-06-0 - 18a -SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) API?LICANT: The University of British Columbia (ii) TITLE OF INVENTION: MOLECULAR DIAGNOSIS OF VIRAL INFECTIONS
(iii) NUMBER OF SEQUENCES: 28 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Smart & Biggar (B) STREET: Box 11560, Vancouver Centre 2200 - 650 West Georgia Street (C) CITY: Vancouver (D) PROVINCE: B.C.
(E) COUNTRY: Canada (F) POSTAL CODE: V6B 4N8 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 05-MAR-98 (C) CLASSIFICATION:
(vii) PR:[OR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/040,207 (13) FILING DATE: 06-MAR-1997 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Smart & Biggar (C) REFERENCE/DOCKET NUMBER: 80021-38 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (604) 682-7295 (B) TELEFAX: (604) 682-0274 (2) INFORMATION FOR SEQ ID NO:l:
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MOLECULAR DIAGNOSIS OF VIRAL INFECTIONS
This invention relates to the detection of the genetic material of RNA pathogens and in particular, multiple RNA
respiratory pathogens.
Laboratory testing to identify disease causing pathogens in clinical specimens is necessary in a variety of circumstances. Test results may be useful for guiding physicians in terms of appropriate patient management and medication, gathering information necessary for epidemiology and public health purposes, and research into mechanisms of infections caused by microorganisms.
Conventional laboratory methods of viral diagnosis such as culturing the pathogen, electron microscopy, immunodetection of the pathogen and serology are subject to significant limitations. Such limitations may be brought about by factors such as poor replication of pathogens in culture, requirement for specialized and expensive equipment (e.g. electron microscopy) and, existence of numerous serotypes of a particular pathogen.
Amplification of genetic material derived from a respiratory pathogen provides a sensitive and specific meanE3 for detecting such pathogens. Reverse transcription (RT) of viral RNA followed by DNA amplification in the CA 02231271 1998-03-0~
polyrnerase chain reaction (PCR) has been shown to be a sens:Ltive diagnostic procedure for detection of several RNA
virus respiratory pathogens: Gilbert, L.L., et al. (1996) Diagnosis of Viral Respiratory Tract Infections in Children by using a Reverse Transcription-PCR Panel; Journal of Clin:Lcal Microbiology 34:140-143. However, such procedures are complex and requi:re a significant length of time to be carr:Led out. This is disadvantageous in a commercial clin:Lcal laboratory setting, particularly in situations where the diagnostic procedure must detect multiple RNA
pathogens. In addition to upper and lower respiratory tracl infections (e.g. common cold, pneumonia), multiple RNA respiratory pathogens may infect a single patient show:Lng symptoms of asthma or exercise-induced bronchoconstricton (EIB).
Summary of Invention This invention provides a method to obtain RNA
pathogen cDNA through reverse transcription (RT) wherein an extract of cells suspected of containing a RNA pathogen is used in a RT reaction in which pooled short oligonucleotides of random sequence are used as a source of RT ~rimer. The RNA pathogen may be a RNA respiratory pathogen.
While the method of this invention may be used to obta:in CDNA from the RNA of any pathogen, the method is CA 02231271 1998-03-0~
particularly suited to the diagnosis of multiple RNA
pathogen infections since the RT reaction does not depend upon use of specific oligonucleotide primers. Rather, the use of random oligomers as primers permits reverse transcription of RNA from multiple sources present in a single sample.
Use of random oligomers as RT primers is known for the dete,-tion of RNA from single RNA pathogen sources; see:
M.H. Vodkin et al. (1994) "PCR - Based Detection of Arboviral RNA from Mosquitoes Homogenized in Detergent", BioTechniques 17:114-116; R.F. Meyer et al. (1991) "Rapid and Sensitive Detection of Foot and Mouth Disease Virus in Tissues by Enzymatic RNA Amplification of the Polymerase Gene", Journal of Virological Methods 34:161-172; and, D.K.
Howe et al . ( 1992) "Use of the Polymerase Chain Reaction for t:he Sensitive Detection of St. Louis Encephalitis Viral RNA", Journal of Virological Methods 36:101-110. Also, use of random oligomers as RT primers has been demonstrated for the l~etection of multiple RNA pathogens from non-cellular sour~es; see: Yu-Li Tsai, et al. (1994) "Detection of Polio Virus, Hepatitis A Virus, and Rotavirus from Sewage and Ocean Water by Triplex Reverse Transcriptase PCR", Applied Envi:ronmental Microbiology 60:2400-2407.
This invention provides a method to obtain RNA
resp:iratory pathogen cDNA through reverse transcription CA 02231271 1998-03-0~
(RT) of genetic material obtained from a respiratory source comprising the steps of:
(a) extracting RNA from cells from a respiratory source; and (b) using RNA from step (a) as a template in a RT
reaction in which pooled short oligonucleotides of random sequence are used as a source of RT primer.
RNA respiratory pathogens detectable by the method of this invention include single stranded RNA viruses of which the following are examples: picoronavirus (rhinoviruses and/or enteroviruses); parainfluenza viruses (PIV);
respiratory syncytial virus (RSV); influenza viruses A, B
and C; and, coronaviruses OC43 and 229E.
Cells from a respiratory source may be cells of any respiratory specimen including nasopharyngeal or tracheal washes and aspirates, bronchoalveolar levage fluid, and lung tissue specimens.
Methods for extracting RNA from cells and cell suspensions are well known in the art, such as the procedures described in: Chomczynski, P. and N. Sacchi (1987) Single-Step Method of RNA Isolation by Acid Guan:idinium Thiocyanate-Phenol-Chloroform Extraction; Anal.
Biochem. 162:156-159. A preferred method of RNA extraction by cell lysis is described below.
CA 02231271 1998-03-0~
Methods for performing RT are well known in the art and commercial RT kits are readily available. However, rather than using viral specific oligonucleotide primers, this method employs a pool of random oligomers as a source of the RT primers. The pool of random oligomers should be assernbled such that as many of the possible permutations of random sequences are provided in the pool. This is most conveniently done by mixing different oligomers having the short:est possible sequence that will function efficiently as a RT primer. Hexamers will efficiently act as RT
primers at normal temperatures (e.g. 37~C) and there are only 4,096 possible permutations. Pools of random hexamers for use as primers are commercially available (e.g Pharmacia, Montreal, Canada).
This invention also provides a method of amplifying DNA derived from two or more sources by PCR in a single samp~Le. For example, the DNA may be cDNA derived from two or more RNA respiratory pathogens. According to this aspect of the invention, DNA derived from different sources is annplified in a single sample using primers specific for two or more pathogens. Preferably, the primers are selected such that the amplified DNA associated with different pathogens will be of such differing lengths as to be d:iscernible following usual separation procedures such as electrophoresis. PCR amplification procedures and reagents are well known in the art. Combination of the above described method of RT using random oligomers with CA 02231271 1998-03-0~
multiple PCR amplification in a single sample provides a great:ly streamlined yet efficient process for obtaining detectable levels of genetic material derived from multiple RNA pathogens.
This invention also provides an efficient method for amplification of DNA, such as cDNA derived from RNA
respi.ratory pathogens r whereby the resulting amplified DNA
may be detected by an immunostaining procedure which does not require the use of radio-labelled probes yet is comparable in sensitivity to the latter procedure. This method facilitates the above described method of multiple PCR amplification in cL single sample since its sensitivity permits the detection of product which is present in a minut:e amount as compared to other amplified product in the samp]e. The method was described by the inventors in: A
Nonradioactive Method for Rapid and Sensitive Detection of Polymerase Chain Reaction Products by use of Bromo-Deoxyuridine (1996) Modern Pathology 9:849-853.
This invention also provides kits comprising reagents for carrying out the aforementioned methods. A kit for performing the above described RT method will comprise a pool of random oligomers for use as RT primers, preferably a pool of random hexamers. When the kit is to be used for carrying out the above described method of multiple PCR
amplification, it will comprise RNA respiratory pathogen speciLfic primers. Such a kit will preferably also comprise CA 02231271 1998-03-0~
a dNTP mixture in which bromo-deoxyuridine (Br-dUTP) is subst:ituted for dTTP. Such a kit would also preferably include an anti-Br-dU antibody and the remainder of a ELISA
system whereby amplified DNA containing Br-dU may be readily detected (e.g. by enzyme-chemiluminescence).
Detailed Description of the Invention Detailed descriptions of embodiments of this invention are cet out in the accompanying examples. Unless otherwise stated, autoclaved plasticware (at least 20 min. on dry heat, 30 psi, 132~C) is used, and aqueous solutions contain 0.1~ diethylpyrocarbonate (DEPC) as an RNAse inhibitor. To prevent inadvertent exogenous RNAse activity from handling of specimens, investigators should wear latex gloves that are changed several times during the course of the procedure.
RNA ~xtraction. Total RNA is extracted from a thawed cell suspension (e.g., nasopharyngeal swabs, bronchoalveolar lavage fluid) by use of the RNeasyTM spin column, lysis and wash buffers (Qiagen Corporation) as follows: a 300 ~L
aliquot of the specimen is added to an autoclaved 1.5 mL
Eppendorf tube, followed by addition of 300 ~L of lysis buffer RLT, 600 uL of 70~ ethanol and mixing by agitation on a vortex. This 1.2 mL sample is applied onto the RNeasyTM spin column and centrifuged for 15 s at 10,000 rpm.
The flowthrough is discarded. Seven hundred ~L of wash CA 02231271 1998-03-0~
buffer RW1 is added to the spin column, the sample is centrifuged as above and the flowthrough is discarded. The spin column is placed in a new 2 mL collection tube. Five hundred ~L of wash buffer RPE is pipeted onto the spin colun~m, and the sample is centrifuged as above, with the flowthrough being discarded. Wash Buffer RPE (500 ~L) is pipet.ed onto the spin column and the sample is centrifuged at full speed for 2 min. The spin column is transferred to a new collection tube and the RNA is eluted by addition of 30 ~l of DEPC-distilled, deionized water and centrifugation at 10,000 rpm for 1 min.
In cases where RNA is extracted from tissue specimens (such as frozen lung tissue) the commercially available TRIZC)LTM reagent may be used (see United States patent No. 'i,346,994.
Reverse transcription (RT). To remove secondary structure from RNA, a 10 ~L aliquot from the RNA sample as prepared above is heated to 68"C for 10 min., then quick-chilled on ice. This linearized RNA is added to an Eppendorf tube that contains 10 ~L of the following aqueous RT reaction solut:ion at room temperature: 10 mM Tris-HCl (pH 8.3);
50 mM KCl, 5 mM MgCl2, 10 mM pooled deoxynucleotide triphosphates (dNTPs),(Pharmacia) 1 ~L of 50 ~M random hexamers to act as "universal" primers for RT, 200 units Moloney murine leukemia virus reverse transcriptase, (Pharmacia) and 20 units of RNAse inhibitor (Pharmacia).
CA 02231271 1998-03-0~
g RT occurs in the closed Eppendorf tube at 37~C for 60 min., after which time the tube is heated at 95~C for 5 min. to inactivate the reverse transcriptase. The specimen is then placed in ice until used for PCR.
5 PCR almplification. PCR amplification is carried out in a 50 IlL, reaction volume containing: all or part of the 20 ~L
aliquot from the RT procedure; 10 mM Tris-HCl, pH 8.3;
50 mM KCl; 5 mM MgCl2; 0.2 mM dNTPs, in which an equimolar concentration of 5-bromo-2-deoxyuridine triphosphate 10 (Br-clUTP; Sigma) is substituted for deoxythymidine triphosphate; 2 ~M of oligonucleotide primers specific to two pathogens and, 2 units of Taq DNA polymerase (Gibco-BRL). Specimens undergo brief centrifugation and are overlayed with mineral oil. PCR cycling conditions on 15 a ROBOCYCLER 40TM (Stratagene) involve 35 cycles, each one consisting of denaturation at 94~C for 1 min., annealing at 55~C for 1 min., and extension at 72~C for 1 min. (10 min.
during the last cycle). Samples undergo electrophoresis on l-l.Ci% agarose gels that contain 0.5 ~g/mL ethidium 20 bromide. This is followed by conventional Southern blott:ing on nylon membranes (e.g. Hybond-NTM membranes, Amersham) and fixation of any PCR products (DNA) by cross-linking under ultraviolet light for 2-5 min.
Immunodetection of PCR products. Nylon membranes are 25 pre-equilibrated in 0.1 M Tris buffered saline (TBS), pH
7.5 :Eor 5 minutes at room temperature and then incubated CA 02231271 1998-03-0~
with blocking buffer (0.1 M TBS, pH 7.5 with 5~ non-fat dry milk powder (Carnation)) at 37~C for 15 min. Mouse anti-bromodeoxyuridine antibody (Boehringer Mannheim) is addecl at a final dilution of 1:1,000 in blocking buffer for a 45 min. incubation at 37~C. After three washes of 5 min.
each in TBS, the nylon membranes are incubated at 37~C for min. with peroxidase-conjugated goat anti-mouse immunoglobulin diluted 1:2,000 in blocking buffer. After three final washes in TBS, enzyme-chemiluminescent (ECL) substrate (Amersham) is applied and the membranes are expo~:ed to x-ray film with regular intensifying screens for a period varying from 15 sec. to 5 min.
Example Children selected from a survey of primary school chilclren in Port Alberni, British Columbia, Canada, were identified as having asthma, EIB or as controls. Age and sex distribution of the study participants is shown in Table 1. A nasal swab specimen was taken from each child from the left inferior nasal turbinate with the swab being rotat:ed once. Each specimen was stored in a cryovial on ice and was subsequently refrigerated at -70~C pending further processing. Positive controls for RT-PCR were prepared from total cellular RNA extracted from virus infected cell cultures grown in M199 minimum essential medium (Gibco BRL) supplemented with 5~ fetal calf serum, L-glutamine and vitamins. All viruses and cell lines were CA 02231271 1998-03-0~
obtained from American Type Culture Collection. HEp-2 cells were infected with Long strain type A RSV; MRC-5 cells were infected with human rhinovirus, type lB; Rhesus monkey kidney (MK2) cells were infected with one of:
picornavirus, PIV type 3, Influenza A Weiss-43, human coronavirus OC43, and human coronavirus 229E. Negative controls included blank reagent mixtures which contained all constituents except a nucleic acid template, or with a nucleic acid template prepared from total cellular RNA extracted from uninfected cell cultures of the appropriate type.
The above described procedures using a RNeasy'M spin column were carried out for RNA extraction. Total RNA was eluted into 30 ~L of 0.1~ DEPC containing distilled water.
Two 10 ~L aliquots of total RNA were incubated for ten minut:es at 70~C to remove secondary structure and were quickly chilled on ice. Reverse transcription accordingto the above described procedures using random hexamers as primers were carried out for each aliquot of linearized RNA.
Five ~L aliquots of cDNA from the RT step were added to separate 45 ~L aliquots of PCR reaction buffer containing 10 mM Tris-HC1 (pH 8.3), 5.0 mM MgC12, 10 mM
pooled dNTPs, 20 U Taq polymerase (GIBCO BRL), and 2 ~M of viru~;-specific oligonucleotide primers as shown in Table 2.
PCR was performed on a Robocycler 40TM (Stratagene, La TABLEI. Age and sex distribution of study pantcipants ASTH~LA E~B CONTROL
(n = 21) (n = 16) (n = 33) Male/Female 12/9 6/10 14/19 Median age i S:D 11.8 + 2.0 11.4 + 2.0 11.4 + 1.8 (years) TABLE2.01igon~s used for viral RT-PCR
P~ K
Virus RegionOligonucleotide Sequence (5' - 3')Product Size (bp) RSV nucleocapsid primer I GCGATGTCTAGGTTAGGAAGAA 410 primer 2 GCTATGTCCTTGGGTAGTAAGCCT
probe TAGCTCCAGAATACAGGCATGACTC
PIV F gene primer I AGAGGTCAATACCAACAACTA 20S
5' nont~nslated primer 2 TAGCAGTATTGAAGTTGGCA
region probe AAAATTCCAAAACAGACCGGC
Picornavirus 'i' nontranslated primer I GCA~-l-l~-l~l-l-l~CCC 380 region of primer 2 CGGACACCCAAAGTAG
rhinovirus IB probe GCATTCAGGGGCCGGAG
In~uenza A matri~ protein primer I CAGAGACTTGAAGAT~l~l-l-lGC 212 primer 2 G~l~-l~lCCATGTTATTTGGATC
probc TCCTGTCACCTCTGACTAAGGGGATTTTG
Coronavirus nucleocapsid primer I GGTACTCCTAAGC~ l~-lCG 370 229E primer 2 TGCACTAGGGTTAATGAAGAGG
probe GACTATCAAACAGCATAGCAGC
Coronavirus nucleocapsid primer l AGGAAGGTCTGCTCCTAATTCC 4so OC43 primer 2 TGCAAAGATGGGGAACTGTGGG
probe GGTCTGGCAAAACTTGGCAAGG
CA 0223l27l l998-03-0 Jolla" CA). For each virus tested by the panel, 35 PCT
cycles were performed, each cycle consisting of 1 min.
denaturation of 94~C, 1 min. annealing at 55~C and 2 min.
extension at 72~C (10 min. during cycle 35). A PCR
experiment consisted of testing 10 patient specimens for one of the six asthma-associated viruses.
After completion of PCR, a 45 ,uL aliquot of each PCR
s amp le un de rwent e lect rop ho re sis o n ethidium-bromide-stained, 1-2~ agarose gels (GIBCO BRL), in TBE buffer that contained 44.5 mM Tris-HC1, 44.5 mM boric acid and 1 mM EDTA, Gels were photographed under ultraviolet light using Polaroid 667 ISO 3000 professional print film, after which RT-PCR products underwent Southern transfer onto nylon membranes, hybridization with the virus-specific,32P-labelled oligonucleotide probes as shown in Table 2, and autoradiography.
CA 0223l27l l998-03-0 Viral RNA was detected in 43/70 (61.4~6) of specimens.
Influenza A viral RNA was detected in higher proportion of specimens from clinically stable asthmatic children and children with EIB, in comparison to asymptomatic controls.
Table 3 summarizes the proportion of subject showing evidence of RNA from each of the viruses examined. Table 4 shows that there were similar proportions of subjects in the three groups who had none, one or multiple viruses detected by the RT-PCR panel. The results reveal a high prevalence picoranviral RNA in the three groups of children. The RT protocol may have yielded more virus related cDNA for PCR amplification since DNA could be synthesized from both the genomic RNA of the virus and from viral mRNA transcripts. The random hexamers acting as primers would permit reverse transcription of both forms of virus specific RNA despite differences in polarity.
Exam~)le 2 cDNA samples resulting from the above described RT
procedures are subjected to duplex PCR amplification using the primers shown in Table 5. In each duplex reaction, a cDNA sample is exposed to primers for two pathogens whereby amplification of cDNA associated with each of the pathogens occurs simultaneously in a single sample. The following scheme is employed in order that the amplification products will differ significantly in size to facilitate elect:rophoretic separation:
CA 02231271 1998-03-0~
(a) picornavirus and parainfluenza virus (PIV);
(b) RSV and influenza A virus;
(c) influenza B and C viruses; and (d) Coronaviruses OC43 and 229E.
Detection of amplification products may be carried out according to the radio labelling procedure of Example 1 using labelled probes as shown in Table 2, or by detection of the incorporation of Br-U in amplified DNA as described above and in Example 3.
Simultaneous detection of DNA respiratory pathogens such as adenovirus and Chlamydia may be carried out.
Suitable PCR primers for the latter organisms are shown in Table 5. The procedure employed may involve either RT of total RNA in a sample followed by amplification of cDNA
derived from both RNA and DNA pathogens. Alternatively, a DNA preparation obtained from the respiratory source may be mixed with a cDNA preparation obtained from the above descr-ibed RT step and the single sample is then subjected to multiple PCR amplification of RNA pathogen cDNA and DNA
pathogen DNA according to the methods described above.
CA 0223l27l l998-03-05 TA~BLE 3. Number (perccntage) of subjects showing positive RT-PCR results for each vi us in the panel.
AST~A EIB CON~OL
(n = 21' (n = 16) (n = 33' RSV ~~ 4' ~ (0) 8 '.4 Pl~ (6) 5 i5, Picornaviru~ (25) ,~4 Tnfll~en7 A ~ (3') ( ) Coronav ~i229E 2 J) (0) _ (6) Coronav,~; OC43 3 ~14) ~ (25) J (15) p = 0.034 in co~palison to asymptom~-i~ controls TABLE 4. Number (percent~ge) of subjects showing viral RNA detected by the RT-PCR panel Number of viruses detected by ASTE~IA EIB CONTROL
Rl -PCR panel (n = 21' (n = 16' (n = 33' vimserl-~c-~d ' 6 3 3 ~' virus ~ ~ct~ 5 3 ~ " ' ' 2 vin~es ~etect~d 5 ~- 5 ~3 0 CA 0223l27l l998-03-05 TABLE 5. PCR Primers Picon~(380~product) 5'CGGACACCCAAAGTAG3' 5'GCACTTCTG m CCCC3' P~a~flu~I~(205~) S'AGAGGTCAATACCAACAACTA3' 5'TAGCAGTATTGAAGTTGGCA3' RSV(410~) 5'GCGATGTCTAGGTTAGGAAGAA3' 5'GCTATGTCCTTGGGTAGTAAGCCT3' ~fluo~aA(212~) S'GCTCTGTCCATGTTATTTGGATC3' S'CAGAGACTTGAAGATGTC~ lGC3' ~flu~aB(36~ ~) S'AGCGTTCCTAGl-l-l-lACTTGCAT3' S'GAAAAATTACACTGTTGGTTCGG3' ~flu~aC(425~) S'GCCAGTAATACCAGCAATCTCG3' S'CCCTAATGTCTTGGAGAAGCCAC3' Coro~n~OC43(450~) 5'TGCAAAGATGG~GAACTGTGGG3' 5'AGGAAGGTCTGCTCCTAATTCC3' C~o~n~229E(370~) 5'TGCACTAGGGTTAATGAAGAGG3' S'GGTACTCCTAAGCCTTCTCG3' Ad~o~n~(300~) S'CAGCACGCCGCGGATGTCAAAGT3' 5'GCCGCAGTGGT~TTACATGCACATC3' Chl~my~a(l45~) 5'GCCTGTAGGGAAT(CG)CAGCT(CG)AA(CT)CA3' S'GTCGAAAACAAAGTC(AT)CC{AG)TAGTA3' CA 0223l27l l998-03-0 Example 3 The above described PCR procedures are carried out using the method described by the inventors at: (1996) Modern Pathology 9: 849-853 in which dTTP is substituted by Br-dUTP in the PCR reaction followed by electrophoresis, Southern Blotting and immunostaining as described above.
Duplex RT-PCR for RSV and influenza A virus in the presence of Br-dUTP was carried out with a constant amount of RSV
and serial 10-fold dilutions of influenza A virus. The results yield a distinct influenza A band (212 bp product) that may be detected in specimens in which 1:1000 of the starting amount of viral RNA was mixed with a large excess of RSV RNA ( 410 bp product).
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 attached claims.
CA 0223l27l l998-06-0 - 18a -SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) API?LICANT: The University of British Columbia (ii) TITLE OF INVENTION: MOLECULAR DIAGNOSIS OF VIRAL INFECTIONS
(iii) NUMBER OF SEQUENCES: 28 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Smart & Biggar (B) STREET: Box 11560, Vancouver Centre 2200 - 650 West Georgia Street (C) CITY: Vancouver (D) PROVINCE: B.C.
(E) COUNTRY: Canada (F) POSTAL CODE: V6B 4N8 (v) COMPUTER READABLE FORM:
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(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 05-MAR-98 (C) CLASSIFICATION:
(vii) PR:[OR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/040,207 (13) FILING DATE: 06-MAR-1997 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Smart & Biggar (C) REFERENCE/DOCKET NUMBER: 80021-38 (ix) TELECOMMUNICATION INFORMATION:
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(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
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(2) INFORMATION FOR SEQ ID NO:18:
CA 0223l27l l998-06-0 - 18f -(i) SE,QUENCE CHARACTERISTICS:
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(2) INFORMA.TION FOR SEQ ID NO:l9:
(i) SE,QUENCE CHARACTERISTICS:
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(i) SEQUENCE CHARACTERISTICS:
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(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
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(2) INFORMATION FOR SEQ ID NO:22:
CA 0223l27l l998-06-0 - 18g -(i) SE,QUENCE CHARACTERISTICS:
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(2) INFORMATION FOR SEQ ID NO:23:
(i) SE,QUENCE CHARACTERISTICS:
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(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
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(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
(2) INFORMATION FOR SEQ ID NO:26:
CA 0223l27l l998-06-0 - 18h -(i) SE'QUENCE CHARACTERISTICS:
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(2) INFORMA.TION FOR SEQ ID NO:27:
(i) SE,QUENCE CHARACTERISTICS:
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(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
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Claims (8)
1. A method to obtain cDNA derived from multiple RNA
respiratory pathogens present in cells from a respiratory source, comprising the steps of:
(a) extracting RNA from the cells;
(b) mixing RNA from step (a) with pooled short oligonucleotides of random sequence in the presence of reverse transcriptase and deoxynucleotide triphosphates to produce cDNA;
(c) mixing cDNA obtained in step (b) with oligonucleotide primers specific to two or more RNA
respiratory pathogens in the presence of deoxynucleotide triphosphates, and DNA polymerase; and (d) performing a polymerase chain reaction by altering conditions so as to repetitively denature, anneal, and extend DNA thereby producing multiple copies of cDNA
obtained in step (b) having sequences hybridizable to said oligonucleotides specific to respiratory pathogens.
respiratory pathogens present in cells from a respiratory source, comprising the steps of:
(a) extracting RNA from the cells;
(b) mixing RNA from step (a) with pooled short oligonucleotides of random sequence in the presence of reverse transcriptase and deoxynucleotide triphosphates to produce cDNA;
(c) mixing cDNA obtained in step (b) with oligonucleotide primers specific to two or more RNA
respiratory pathogens in the presence of deoxynucleotide triphosphates, and DNA polymerase; and (d) performing a polymerase chain reaction by altering conditions so as to repetitively denature, anneal, and extend DNA thereby producing multiple copies of cDNA
obtained in step (b) having sequences hybridizable to said oligonucleotides specific to respiratory pathogens.
2. The method of claim 1 wherein the deoxynucleotide triphosphates in step (c) comprise 5-bromo-2-deoxyuridine triphosphate and the method includes the additional step of detecting bromodeoxyuridine containing DNA with a anti-bromodeoxyuridine antibody.
3. The method of claim 1 or 2 wherein the pooled short oligonucleotides of random sequence are hexamers.
4. A kit comprising a pool of short oligonucleotides of random sequence for use in reverse transcription; and oligonucleotides hybridizable to unique cDNA sequences corresponding to RNA of two or more respiratory pathogen.
5. The kit of claim 4 wherein the short oligonucleotides of random sequence are hexamers.
6. The kit of claim 4 or 5 further comprising a reverse transcriptase and a DNA polymerase.
7. The kit of claim 4 5 or 6 further comprising deoxynucleotide triphosphates for use with the reverse transcriptase and deoxynucloetide triphosphates which comprise 5-bromo-2-deoxyuridine triphosphate for use with the DNA polymerase.
8. The use of pooled short oligonucleotide of random sequence in a reverse transcription reaction to obtain RNA
pathogen cDNA from RNA obtained from an extract of cells suspected of containing RNA from two or more RNA pathogens.
pathogen cDNA from RNA obtained from an extract of cells suspected of containing RNA from two or more RNA pathogens.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US4020797P | 1997-03-06 | 1997-03-06 | |
| US60/040,207 | 1997-03-06 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2231271A1 true CA2231271A1 (en) | 1998-09-06 |
Family
ID=21909724
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2231271 Abandoned CA2231271A1 (en) | 1997-03-06 | 1998-03-05 | Molecular diagnosis of viral infections |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2231271A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1877418A2 (en) * | 2005-05-06 | 2008-01-16 | Gen-Probe Incorporated | Compositions and assays to detect influenza virus a and b nucleic acids |
| WO2009009900A1 (en) * | 2007-07-17 | 2009-01-22 | Universite Laval | Nucleic acid sequences for the amplification and detection of respiratory viruses |
| CN102719564A (en) * | 2012-06-25 | 2012-10-10 | 广西壮族自治区兽医研究所 | Triple polymerase chain reaction (PCR) kit for duck hepatitis virus type I, duck circoviruses and Muscovy duckling parvovirosis and application of triple PCR kit |
-
1998
- 1998-03-05 CA CA 2231271 patent/CA2231271A1/en not_active Abandoned
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1877418A2 (en) * | 2005-05-06 | 2008-01-16 | Gen-Probe Incorporated | Compositions and assays to detect influenza virus a and b nucleic acids |
| EP1877418A4 (en) * | 2005-05-06 | 2009-12-09 | Gen Probe Inc | Compositions and assays to detect influenza virus a and b nucleic acids |
| US8124335B2 (en) | 2005-05-06 | 2012-02-28 | Gen-Probe Incorporated | Compositions and assays to detect influenza virus A and B nucleic acids |
| US8338095B2 (en) | 2005-05-06 | 2012-12-25 | Gen-Probe Incorporated | Compositions and assays to detect influenza virus A and B nucleic acids |
| WO2009009900A1 (en) * | 2007-07-17 | 2009-01-22 | Universite Laval | Nucleic acid sequences for the amplification and detection of respiratory viruses |
| CN102719564A (en) * | 2012-06-25 | 2012-10-10 | 广西壮族自治区兽医研究所 | Triple polymerase chain reaction (PCR) kit for duck hepatitis virus type I, duck circoviruses and Muscovy duckling parvovirosis and application of triple PCR kit |
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