CN102433393A - Primers, probe and method for detecting various respiratory viruses - Google Patents
Primers, probe and method for detecting various respiratory viruses Download PDFInfo
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Abstract
The invention provides a primer combination for detecting various respiratory viruses, a specific probe combination for detecting various respiratory viruses, a method using the primers and probe to detect various respiratory viruses and a kit comprising the primer combination and the probe combination. In the invention, the mixed solution of the specific primers and the probe with fluorescent mark is utilized to carry out PCR (Polymerase Chain Reaction) amplification on batch samples, and then microsatellite analysis is performed, so as to analyze a great quantity of samples at one time and detect various respiratory viruses simultaneously, thus significantly improving efficiency and realizing high sensitivity. The kit can be popularized and used clinically, and has wide application prospect.
Description
Technical field
The present invention relates to the viral diagnosis technical field, specifically, is a kind of primer, probe and method that detects various respiratory road virus.
Background technology
Respirovirus is one type can invade that respiratory tract causes the respiratory tract local patholoic change or be portal entry with the respiratory tract only, mainly causes the virus of the outer histoorgan pathology of respiratory tract.According to statistics, 90% above ARI is caused by virus, and its infectivity and pathogenic very strong, generally can directly propagate interpersonal through the saliva spittle.Therefore, must strengthen diagnosis, with active treatment, prevent to propagate to Respirovirus.
Respirovirus comprises the influenza virus in the orthomyxoviridae family (Orthomyxoviridae); Some viruses in parainfluenza virus in the Paramyxoviridae (Paramyxoviridae), respiratory syncytial virus, Measles virus, mumps virus and other Viraceae are like adenovirus, rubella virus, rhinovirus, coronavirus and reovirus etc.Common clinically Respirovirus comprises that respiratory syncytial virus A (RSVA) and respiratory tract close these 15 kinds of spore virus B (RSVB), influenza virus A type and influenza virus Type B (FluA and FluB), rhinovirus (HRV), parainfluenza virus 1-4 type (PIVl-PIV4), enterovirus (HEV), adenovirus (ADV), coronavirus (OC43,229E/NL63), human metapneumovirus (MPV) and human bocavirus (HBoV).Have viral analytical method, serodiagnosis, immunofluorescence technique and nucleic acid hybridization, PCR or sequential analysis that its detection method is commonly used detect the method for viral nucleic acid.And in the middle of these methods, nucleic acid detection method is highly sensitive because of it, high specificity, detection speed are superior to additive method soon.But need to detect sample more for a long time when clinical, for nucleic acid detection method, as only adopting to the single reaction system of single virus, then workload is very big, wastes time and energy and wastes resource.
Chinese patent document CN 201010542857.8; November 12 2010 applying date; A kind of method and primer and probe that detects various respiratory road virus disclosed; This invention to adenovirus, human metapneumovirus, influenza virus A type, influenza virus B type, respiratory syncytial virus, bocavirus, rhinovirus, coronavirus (HKU1, NL63 and SARS), parainfluenza virus (I type, II type, III type and IV type) in totally 14 the Respirovirus nucleotide sequence analyze; Design corresponding reverse transcription primer, PCR primer and specific probe; With the method amplification specific gene degree of bias of reverse transcription and multiple asymmetric PCR, adopt fluorescence-encoded micro-beads group and the pcr amplification product incubation hybridization of liquid-phase chip technology with coupling idol virus-specific probe, use Bio-Plex then
TM200 detect.Though this invention flux is bigger, has accelerated detection speed to a certain extent, this method at first need be with nucleic probe and microballoon coupling; Also need the pcr amplification product and the nucleic probe microballoon group of sample be hybridized at last, mark; Program is comparatively loaded down with trivial details, and in addition, this method can not be diagnosed viruses such as coronavirus OC43,229E/NL63; Therefore, need badly and a kind ofly can detect viral, more quick, economic, the effective detection method in various respiratory road simultaneously.
Summary of the invention
The objective of the invention is to deficiency of the prior art, a kind of combination of primers that detects various respiratory road virus is provided.
One purpose more of the present invention is that a kind of probe combinations that detects various respiratory road virus is provided.
Another purpose of the present invention is that a kind of method that detects various respiratory road virus is provided.
The 4th purpose of the present invention is that a kind of test kit that detects various respiratory road virus is provided.
The 5th purpose of the present invention is that the purposes of a kind of above-mentioned primer and probe is provided.
For realizing above-mentioned purpose, the technical scheme that the present invention takes is:
A kind of combination of primers that detects various respiratory road virus, described combination of primers is a pair of or several to forming by following primer centering: SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30.
Described combination of primers is:
(a) combination of primers I:SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8 and SEQ ID NO.9-10; Or
(b) combination of primers II:SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18 and SEQ ID NO.19-20; Or
(c) combination of primers III:SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28 and SEQ ID NO.29-30.
For realizing above-mentioned second purpose, the technical scheme that the present invention takes is:
A kind of probe combinations that detects various respiratory road virus, described probe combinations is made up of in the probe of nucleotide sequence shown in SEQ ID NO.31 ~ 45 one or more.
Described probe combinations is:
(a) probe combinations I:SEQ ID NO.31 ~ 35; Or
(b) probe combinations II:SEQ ID NO.36 ~ 40; Or
(c) probe combinations III:SEQ ID NO.41 ~ 45.
5 ' end of the probe of described nucleotide sequence shown in SEQ ID NO.31 ~ 35 is modified by the 6-Fluoresceincarboxylic acid; 5 ' end of the probe of described nucleotide sequence shown in SEQ ID NO.36 ~ 40 is modified by chlordene resorcinolphthalein phosphoramidate; 5 ' the end of described nucleotide sequence shown in SEQ ID NO.41 ~ 45 modified by carboxyl-X-rhodamine succinimide ester.
For realizing above-mentioned the 3rd purpose, the technical scheme that the present invention takes is:
A kind of method that detects various respiratory road virus; It is that reverse transcription product with the respiratory mucus sample is a template; Use two or more the combination of primers of Respirovirus of specific amplification; And corresponding specific probe is combined into the performing PCR reaction, is that sample carries out little satellite and detects again with the reaction product.
Described method is that the reverse transcription product with the respiratory mucus sample is a template, uses
(a) aforesaid combination of primers I and aforesaid probe combinations I; Or
(b) aforesaid combination of primers II and aforesaid probe combinations II; Or
(c) aforesaid combination of primers III and aforesaid probe combinations III carry out the PCR reaction, are that sample carries out little satellite detection again with the reaction product.
For realizing above-mentioned the 4th purpose, the technical scheme that the present invention takes is:
A kind of test kit that detects various respiratory road virus, it comprises following reagent:
(a) primer: aforesaid combination of primers I and/or combination of primers II and/or combination of primers III;
(b) probe: aforesaid probe combinations I and/or probe combinations II and/or probe combinations III.
Said test kit also comprises dNTPs, PCR reaction solution and archaeal dna polymerase.
For realizing above-mentioned the 5th purpose, the technical scheme that the present invention takes is:
Nucleotides sequence shown in SEQ ID NO.1 ~ 45 is listed in the application that detects in the Respirovirus.
The invention has the advantages that:
1, the high specificity of primer of the present invention and probe is highly sensitive, can accurately detect various respiratory road virus;
2, the present invention adopts Auele Specific Primer and has the mixing solutions of fluorescence labeling probe; The batch sample is carried out pcr amplification, carry out little satellite (STR) analysis again, the disposable great amount of samples of analyzing; Simultaneously various respiratory road virus is detected; Reduce operation steps, save resource, raise the efficiency;
3, test kit of the present invention can be widely used in the detection of clinical Respirovirus, has broad application prospects.
Description of drawings
Accompanying drawing 1 is that the fluorescent mark of sample one is the collection of illustrative plates of 6 FAM.
Accompanying drawing 2 is that the fluorescent mark of sample one is the collection of illustrative plates of HEX.
Accompanying drawing 3 is that the fluorescent mark of sample two is the collection of illustrative plates of 6 FAM.
Accompanying drawing 4 is that sample two fluorescent marks are the collection of illustrative plates of ROX.
Accompanying drawing 5 is that the fluorescent mark of sample three is the collection of illustrative plates of HEX.
Accompanying drawing 6 is that the fluorescent mark of sample four is the collection of illustrative plates of 6 FAM.
Accompanying drawing 7 is that the fluorescent mark of sample five is the collection of illustrative plates of HEX.
Accompanying drawing 8 is that the fluorescent mark of sample six is the collection of illustrative plates of HEX.
Accompanying drawing 9 is that the fluorescent mark of sample seven is the collection of illustrative plates of ROX.
Accompanying drawing 10 is that the fluorescent mark of sample eight is the collection of illustrative plates of ROX.
Accompanying drawing 11 is that the fluorescent mark of sample nine is the collection of illustrative plates of ROX.
Embodiment
Below in conjunction with accompanying drawing embodiment provided by the invention is elaborated.
Embodiment 1
Use primer of the present invention and probe in detecting various respiratory road virus
1 materials and methods
1.1 sample source
Have heating, cough, runny nose and pharyngalgia etc. through cooperative relationship collects that on June 5th, 7 days 1 June in 2009, fever clinic of Xinhua Hospital Attached to Medical School, Shanghai Jiaotong Univ. went to a doctor are distributed 0-1 year infant 62 examples of upper respiratory tract infection symptoms; Detect; Extract 9 examples, be designated as sample one ~ sample nine respectively.
1.2 the extraction of collection of specimens and viral nucleic acid and reverse transcription
Use throat swab, gather infant upper respiratory tract mucus.Throat swab is put 4ml transhipment liquid (including Hanks liquid and 10% glycerine), and 4 ℃ are transported to the laboratory, and-70 ℃ of preservations are subsequent use.Extract the virus total RNA/DNA in the sample with viral RNA/DNA extraction test kit (purchasing the company in Promega), become cDNA to the reverse transcription of RNA viruses elder generation ,-20 ℃ of preservations are subsequent use.
1.3 synthetic pcr amplification primer and probe
Utilize bioinformation to gain knowledge and associated biomolecule information science software such as DNAstar; The nucleotide sequence that the respiratory syncytial virus A (RSVA) that can retrieve in the Genbank DB and respiratory tract is closed spore virus B (RSVB), influenza virus A type and influenza virus Type B (FluA and FluB), rhinovirus (HRV), parainfluenza virus 1-4 type (PIVl-PIV4), enterovirus (HEV), adenovirus (ADV), coronavirus (OC43,229E/NL63), human metapneumovirus (MPV) and human bocavirus (HBoV) carries out the virus gene sequence compare of analysis; The specific sequence of selected various viruses is designed segmental degenerate pcr primer of corresponding specific gene (seeing table 1) and specific dna probe (seeing table 2) to various viruses.All degenerated primers and probe are synthetic by TaKaRa company.
The purpose fragment length of table 1 primer sequence and amplification
Annotate: the primer title is with the virus name name of correspondence; F1 represents upstream primer, and R1 represents downstream primer; Y=C/T, S=G/C, R=A/G, B=C/G/T.
Table 2 probe sequence
| Title | Probe sequence (5 ' → 3 ') | Fluorescent mark | SEQ ID NO. |
| ADV-piva2 | GGAYACGGSCTCATGGRGACCAGAGG | 5'6-FAM | 31 |
| 229E-s | TGCTTAAGTAGTATACYTCTGCTTST | 5'6-FAM | 32 |
| PIV2-hn | GTGACTGAACAGCYYTTGCGRTTG | 5'6-FAM | 33 |
| PIV3-hn | TACAASGTATASCATCATTATACCB | 5'6-FAM | 34 |
| PIV1-hn | TCCTGCAGCTATTACRRAACATGAB | 5'6-FAM | 35 |
| OC43-pol | TRRCAGACAACABYATCATCACTC | 5' HEX | 36 |
| hRV-5' utr | AAGCACTBCBGTTTCSCCGGT | 5' HEX | 37 |
| RSVA-f | GAATTTTABCARTCARCATGCAGT | 5' HEX | 38 |
| FluA-m1 | CTGYTGTATRBGAGGCCCAT | 5' HEX | 39 |
| RSVB-f | GASAAGCACCACAGTATATGARCBACA | 5' HEX | 40 |
| HBoV-ns1 | CATTAARCACTBTTCCCASCRAA | 5' ROX | 41 |
| FluB-pb1 | TTACATGTTSGGBAAAAGTCSTTTA | 5' ROX | 42 |
| hMPV-f | GCGGCARTTTTYAGAYARTGC | 5' ROX | 43 |
| PIV4-p | CTGTBATTTTRAGTGCRTCTA | 5' ROX | 44 |
| hEV-pol | CGGCCCCTBAATSCRGCTAA | 5' ROX | 45 |
Annotate: the probe title is with the virus name name of correspondence; The probe sequence of every kind of virus promptly adds the fluorescent mark of the correspondence shown in the table at this viral upstream primer 5' end; 6 Fluoresceincarboxylic acid (6-Carboxyfluorescein; 6 FAM) the look fluorescence that turns blue, chlordene resorcinolphthalein phosphoramidate (Hexachlor, HEX) green-emitting fluorescence; Carboxyl-X-rhodamine succinimide ester (Carboxy-X-rhodamine, ROX) rubescent look fluorescence; Y=C/T, S=G/C, R=A/G, B=C/G/T.
1.4 prepare to mix PCR primer working fluid
(1) each PCR primer of synthetic and probe are mixed with the storage liquid of 100 μ mol/L respectively with distilled water;
(2) with the pairing of primer and probe and be divided into three groups; Every group of each 5 pairs of primers and probe; First group is combination of primers I and probe combinations I: PCR primer and each 5 μ l of probe storage liquid of promptly getting virus of A DV, 229E/NL63, PIV2, PIV3, PIV1 correspondence respectively join in the same 1.5ml Eppendorf pipe, and the distilled water that adds 100 μ l again is and mixes PCR primer working fluid I; Second group is combination of primers II and probe combinations II: PCR primer and each 5 μ l of probe storage liquid of getting virus O C43, HRV, RSVA, FluA, RSVB correspondence respectively join in the same 1.5ml Eppendorf pipe, and the distilled water that adds 100 μ l again is and mixes PCR primer working fluid II; The 3rd group is combination of primers III and probe combinations III: PCR primer and each 5 μ l of probe storage liquid of getting viral HBoV, FluB, MPV, PIV4, HEV correspondence respectively join in the same 1.5ml Eppendorf pipe, and the distilled water that adds 100 μ l again is and mixes PCR primer working fluid III.
1.5 pcr amplification reaction:
(1) PCR reaction system: the pcr amplification reaction system is 20 μ l, comprising 10 * PCR Buffer (Mg
2+Plus) 2 μ l; DNTPs (2.5mol/L) 2 μ l, rTaq enzyme (5U/ μ l) 0.1 μ l mixes PCR primer working fluid I (or mix PCR primer working fluid II or mix PCR primer working fluid III) 2 μ l; Sample reverse transcription product (cDNA/DNA) 1 μ l mends ddH
2O12.9 μ l to final volume be 20 μ l;
(2) PCR response procedures: 94 ℃ of 15min → 94 ℃ 0.5min, 60 ℃ of 1.5min, 72 ℃ of 1.5min (40 circulations) → 72 ℃ of 10min → 8 ℃ insulations.
1.6 STR detects
(1) gets 96 hole Sptting plates, use marking pen to indicate plate name, experiment date;
(2) make electronic edition STR and detect table, and the machine table is gone up in generation automatically;
(3) use continuous sample injector, draw the mixture (ROX500 and LIZ500 are mark in the molecular weight) of 990 μ l HIDI and 10 μ l ROX500 or LIZ500, join in the 96 hole Sptting plates every hole 10 μ l;
(4) 96 orifice plates are placed dull and stereotyped whizzer, centrifugal 500g promptly stops;
(5) use 12 rows, the 10 μ L volley of rifle fires, contrast STR detects table, in the corresponding hole of 96 orifice plates, adds the sample of 50pg, i.e. step 1.5 amplification PCR products;
(6) 96 orifice plates are placed dull and stereotyped whizzer, centrifugal 500g promptly stops;
(7) use shrouding film phonograph seal 96 orifice plates, vibration places dull and stereotyped whizzer with 96 orifice plates, and centrifugal 500g promptly stops;
(8) place the PCR appearance, the sex change program is 98 ℃, 5min, and the heat hot lid does not place cool quickly on the mixture of ice and water with 96 orifice plates after the EP immediately;
(9) 96 orifice plates are placed dull and stereotyped whizzer, centrifugal 2000g promptly stops;
(10) use sequenator ABI3730xl to detect the STR sample.
2 results
2.1 the STR of sample one detects collection of illustrative plates
Fig. 1 is the collection of illustrative plates of 6 FAM for the fluorescent mark of sample one, and wherein ADV and PIV3 base number are respectively 534bp and 189bp; Fig. 2 is the collection of illustrative plates of HEX for the fluorescent mark of sample one, and wherein HRV base number is 394bp; The fluorescent mark of sample one is that the collection of illustrative plates of ROX is negative.The result shows that sample one is ADV, PIV3, the HRV positive, and other viruses are negative.
2.2 the STR of sample two detects collection of illustrative plates
Fig. 3 is the collection of illustrative plates of 6 FAM for the fluorescent mark of sample two, and wherein PIV1 and PIV2 base number are respectively 153bp and 264bp; The fluorescent mark of sample two is that the collection of illustrative plates of HEX is negative; Fig. 4 is the collection of illustrative plates of ROX for the fluorescent mark of sample two, and wherein HBoV base number is 579bp.The result shows that sample two is PIV1, PIV2, the HBoV positive, and other viruses are negative.
2.3 the STR of sample three detects collection of illustrative plates
Fig. 5 is the collection of illustrative plates of HEX for the fluorescent mark of sample three, and FluA and RSVB base number are respectively 206bp and 155bp; The fluorescent mark of sample three is that the collection of illustrative plates result of 6 FAM and ROX is all negative.The result shows that sample three is that FluA, RSVB are positive, and other viruses are negative.
2.4 the STR of sample four detects collection of illustrative plates
Fig. 6 is the collection of illustrative plates of 6 FAM for fluorescent mark, and wherein 229E/NL63 and PIV2 base number are respectively 375bp and 264bp; Fluorescent mark is that the collection of illustrative plates of HEX and ROX is negative.The result shows that sample four is that 229E/NL63, PIV2 are positive, and other viruses are negative.
2.5 the STR of sample five detects collection of illustrative plates
Fig. 7 is the collection of illustrative plates of HEX for fluorescent mark, and wherein RSVA base number is 269bp; Fluorescent mark is that the collection of illustrative plates of 6 FAM and ROX is negative.The result shows that sample five is that RSVA is positive, and other viruses are negative.
2.6 the STR of sample six detects collection of illustrative plates
Fig. 8 is the collection of illustrative plates of HEX for fluorescent mark, and wherein OC43 base number is 578bp; Fluorescent mark is that the collection of illustrative plates of 6 FAM and ROX is negative.The result shows that sample six is that OC43 is positive, and other viruses are negative.
2.7 the STR of sample seven detects collection of illustrative plates
Fig. 9 is the collection of illustrative plates of ROX for fluorescent mark, and wherein FluB and HEV base number are respectively 455bp and 194bp; Fluorescent mark is that the collection of illustrative plates of 6 FAM and HEX is negative.The result shows that sample seven is that FluB, HEV are positive, and other viruses are negative.
2.8 the STR of sample eight detects collection of illustrative plates
Figure 10 is the collection of illustrative plates of ROX for fluorescent mark, and wherein MPV base number is 351bp; Fluorescent mark is that the collection of illustrative plates of 6 FAM and HEX is negative.The result shows that sample eight is that MPV is positive, and other viruses are negative.
2.9 the STR of sample nine detects collection of illustrative plates
Figure 11 is the collection of illustrative plates of ROX for fluorescent mark, and wherein PIV4 base number is 249bp; Fluorescent mark is that the collection of illustrative plates of 6 FAM and HEX is negative.The result shows that sample nine is that PIV4 is positive, and other viruses are negative.
Embodiment 2 test kits one of the present invention
Contain following reagent and material in the box:
1. primer solution (100 μ mol/L): consist of primer to SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10
2. probe solution (100 μ mol/L): consist of probe SEQ ID NO.31 ~ 35
3.?dNTPs（2.5mol/L）
4.?10×PCR?Buffer（Mg
2+?Plus）
5. rTaq enzyme (5U/ μ l)
Contain following reagent and material in the box:
1. primer solution (100 μ mol/L): consist of primer to SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20
2. probe solution (100 μ mol/L): consist of probe SEQ ID NO.36 ~ 40
3.?dNTPs（2.5mol/L）
4.?10×PCR?Buffer（Mg
2+?Plus）
5. rTaq enzyme (5U/ μ l)
Embodiment 4 test kits three of the present invention
Contain following reagent and material in the box:
1. primer solution (100 μ mol/L): consist of primer to SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30
2. probe solution (100 μ mol/L): consist of probe SEQ ID NO.41 ~ 45
3.?dNTPs（2.5mol/L）
4.?10×PCR?Buffer（Mg
2+?Plus）
5. rTaq enzyme (5U/ μ l)
Embodiment 5 test kits four of the present invention
Contain following reagent and material in the box:
1. primer solution I (100 μ mol/L): consist of primer to SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30
2. primer solution II (100 μ mol/L): consist of primer to SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20
3. probe solution I (100 μ mol/L): consist of probe SEQ ID NO.31 ~ 35
4. probe solution II (100 μ mol/L): consist of probe SEQ ID NO.36 ~ 40
5.?dNTPs（2.5mol/L）
6.?10×PCR?Buffer（Mg
2+?Plus）
7. rTaq enzyme (5U/ μ l)
Embodiment 6 test kits five of the present invention
Contain following reagent and material in the box:
1. primer solution I (100 μ mol/L): consist of primer to SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30
2. primer solution II (100 μ mol/L): consist of primer to SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20
3. primer solution III (100 μ mol/L): consist of primer to SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30
4. probe solution I (100 μ mol/L): consist of probe SEQ ID NO.31 ~ 35
5. probe solution II (100 μ mol/L): consist of probe SEQ ID NO.36 ~ 40
6. probe solution III (100 μ mol/L): consist of probe SEQ ID NO.41 ~ 45
7.?dNTPs（2.5mol/L）
8.?10×PCR?Buffer（Mg
2+?Plus）
9. rTaq enzyme (5U/ μ l)
Embodiment 7 test kits six of the present invention
Contain following reagent and material in the box:
1. primer solution (100 μ mol/L): consist of primer to SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10
2. probe solution (100 μ mol/L): consist of probe SEQ ID NO.31 ~ 35
Contain following reagent and material in the box:
1. primer solution I (100 μ mol/L): consist of primer to SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30
2. primer solution III (100 μ mol/L): consist of primer to SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30
3. probe solution I (100 μ mol/L): consist of probe SEQ ID NO.31 ~ 35
4. probe solution III (100 μ mol/L): consist of probe SEQ ID NO.41 ~ 45
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; Can also make some improvement and replenish, these improvement and replenish and also should be regarded as protection scope of the present invention.
SEQUENCE?LISTING
< 110>Shanghai City public health clinical center
< 120>a kind of primer, probe and method that detects various respiratory road virus
<130> \
<160> 45
<170> PatentIn?version?3.3
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< 223>primer
<400> 18
ctcaccstgc?ssagtgagcg?gg 22
<210> 19
<211> 27
<212> DNA
<213> artificial?sequence
<220>
< 223>primer
<400> 19
gasaagcacc?acagtatatg?arcbaca 27
<210> 20
<211> 26
<212> DNA
<213> artificial?sequence
<220>
< 223>primer
<400> 20
aaggtstaba?actttggatr?cagcta 26
<210> 21
<211> 23
<212> DNA
<213> artificial?sequence
<220>
< 223>primer
<400> 21
cattaarcac?tbttcccasc?raa 23
<210> 22
<211> 19
<212> DNA
<213> artificial?sequence
<220>
< 223>primer
<400> 22
gacgtsstcc?gbgggraca 19
<210> 23
<211> 25
<212> DNA
<213> artificial?sequence
<220>
< 223>primer
<400> 23
ttacatgtts?ggbaaaagtc?sttta 25
<210> 24
<211> 24
<212> DNA
<213> artificial?sequence
<220>
< 223>primer
<400> 24
ttgsctrtga?csgaarggat?aacc 24
<210> 25
<211> 21
<212> DNA
<213> artificial?sequence
<220>
< 223>primer
<400> 25
gcggcarttt?tyagayartg?c 21
<210> 26
<211> 25
<212> DNA
<213> artificial?sequence
<220>
< 223>primer
<400> 26
gggtagtaar?cabtggatcc?tgcat 25
<210> 27
<211> 21
<212> DNA
<213> artificial?sequence
<220>
< 223>primer
<400> 27
ctgtbatttt?ragtgcrtct?a 21
<210> 28
<211> 24
<212> DNA
<213> artificial?sequence
<220>
< 223>primer
<400> 28
cctratsaca?acaatsaraa?taat 24
<210> 29
<211> 20
<212> DNA
<213> artificial?sequence
<220>
< 223>primer
<400> 29
cggcccctba?atscrgctaa 20
<210> 30
<211> 18
<212> DNA
<213> artificial?sequence
<220>
< 223>primer
<400> 30
ggrtgbccaa?tscratag 18
<210> 31
<211> 26
<212> DNA
<213> artificial?sequence
<220>
< 223>probe
<400> 31
ggayacggsc?tcatggrgac?cagagg 26
<210> 32
<211> 26
<212> DNA
<213> artificial?sequence
<220>
< 223>probe
<400> 32
tgcttaagta?gtatacytct?gcttst 26
<210> 33
<211> 24
<212> DNA
<213> artificial?sequence
<220>
< 223>probe
<400> 33
gtgactgaac?agcyyttgcg?rttg 24
<210> 34
<211> 25
<212> DNA
<213> artificial?sequence
<220>
< 223>probe
<400> 34
tacaasgtat?ascatcatta?taccb 25
<210> 35
<211> 25
<212> DNA
<213> artificial?sequence
<220>
< 223>probe
<400> 35
tcctgcagct?attacrraac?atgab 25
<210> 36
<211> 24
<212> DNA
<213> artificial?sequence
<220>
< 223>probe
<400> 36
trrcagacaa?cabyatcatc?actc 24
<210> 37
<211> 21
<212> DNA
<213> artificial?sequence
<220>
< 223>probe
<400> 37
aagcactbcb?gtttcsccgg?t 21
<210> 38
<211> 24
<212> DNA
<213> artificial?sequence
<220>
< 223>probe
<400> 38
gaattttabc?artcarcatg?cagt 24
<210> 39
<211> 20
<212> DNA
<213> artificial?sequence
<220>
< 223>probe
<400> 39
ctgytgtatr?bgaggcccat 20
<210> 40
<211> 27
<212> DNA
<213> artificial?sequence
<220>
< 223>probe
<400> 40
gasaagcacc?acagtatatg?arcbaca 27
<210> 41
<211> 23
<212> DNA
<213> artificial?sequence
<220>
< 223>probe
<400> 41
cattaarcac?tbttcccasc?raa 23
<210> 42
<211> 25
<212> DNA
<213> artificial?sequence
<220>
< 223>probe
<400> 42
ttacatgtts?ggbaaaagtc?sttta 25
<210> 43
<211> 21
<212> DNA
<213> artificial?sequence
<220>
< 223>probe
<400> 43
gcggcarttt?tyagayartg?c 21
<210> 44
<211> 21
<212> DNA
<213> artificial?sequence
<220>
< 223>probe
<400> 44
ctgtbatttt?ragtgcrtct?a 21
<210> 45
<211> 20
<212> DNA
<213> artificial?sequence
<220>
< 223>probe
<400> 45
cggcccctba?atscrgctaa 20
Claims (10)
1. one kind is detected the viral combination of primers in various respiratory road; It is characterized in that described combination of primers is a pair of or several to forming by following primer centering: SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30.
2. combination of primers according to claim 1 is characterized in that, described combination of primers is:
(a) combination of primers I:SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8 and SEQ ID NO.9-10; Or
(b) combination of primers II:SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18 and SEQ ID NO.19-20; Or
(c) combination of primers III:SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28 and SEQ ID NO.29-30.
3. a probe combinations that detects various respiratory road virus is characterized in that, described probe combinations is made up of in the probe of nucleotide sequence shown in SEQ ID NO.31 ~ 45 one or more.
4. probe combinations according to claim 3 is characterized in that, described probe combinations is:
(a) probe combinations I:SEQ ID NO.31 ~ 35; Or
(b) probe combinations II:SEQ ID NO.36 ~ 40; Or
(c) probe combinations III:SEQ ID NO.41 ~ 45.
5. according to claim 3 or 4 described probe combinations, it is characterized in that 5 ' end of the probe of described nucleotide sequence shown in SEQ ID NO.31 ~ 35 is modified by the 6-Fluoresceincarboxylic acid; 5 ' end of the probe of described nucleotide sequence shown in SEQ ID NO.36 ~ 40 is modified by chlordene resorcinolphthalein phosphoramidate; 5 ' the end of described nucleotide sequence shown in SEQ ID NO.41 ~ 45 modified by carboxyl-X-rhodamine succinimide ester.
6. one kind is detected the viral method in various respiratory road; It is characterized in that; It is that reverse transcription product with the respiratory mucus sample is a template; Use two or more the combination of primers of Respirovirus of specific amplification, and corresponding specific probe being combined into the performing PCR reaction, is that sample carries out little satellite and detects again with the reaction product.
7. method according to claim 6 is characterized in that, it is that reverse transcription product with the respiratory mucus sample is a template, uses
(a) combination of primers I as claimed in claim 2 and probe combinations I as claimed in claim 4; Or
(b) combination of primers II as claimed in claim 2 and probe combinations II as claimed in claim 4; Or
(c) combination of primers III as claimed in claim 2 and probe combinations III as claimed in claim 4 carry out the PCR reaction, are that sample carries out little satellite detection again with the reaction product.
8. one kind is detected the viral test kit in various respiratory road, it is characterized in that it comprises following reagent:
(a) primer: combination of primers I as claimed in claim 2 and/or combination of primers II and/or combination of primers III;
(b) probe: probe combinations I as claimed in claim 4 and/or probe combinations II and/or probe combinations III.
9. test kit according to claim 8 is characterized in that it also comprises dNTPs, PCR reaction solution and archaeal dna polymerase.
10.SEQ the nucleotides sequence shown in ID NO.1 ~ 45 is listed in the application that detects in the Respirovirus.
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| CN201310353132.8A Division CN103451317B (en) | 2011-12-15 | 2011-12-15 | Primers and probes for simultaneously detecting five respiratory tract viruses and application thereof |
| CN201310353152.5A Division CN103451318B (en) | 2011-12-15 | 2011-12-15 | Kit for simultaneously detecting multiple respiratory tract viruses and application thereof |
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Cited By (9)
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| CN103993101A (en) * | 2014-03-07 | 2014-08-20 | 崔淑娟 | Method and kit for simultaneous detection of human coronavirus 229E, OC43 and NL63 |
| CN104059995A (en) * | 2014-06-06 | 2014-09-24 | 深圳市疾病预防控制中心 | Kit and method for simultaneously detecting respiratory syncytial virus, adenovirus and human bocavirus |
| CN104846121A (en) * | 2015-04-18 | 2015-08-19 | 湖北创瑞生物科技有限公司 | Virus triple fluorescence quantitative RT-PCR detection method and virus triple fluorescence quantitative RT-PCR detection kit |
| CN105543414A (en) * | 2016-01-22 | 2016-05-04 | 广州医科大学附属第一医院 | Respiratory syncytial virus A/B subtype multiplex fluorescence quantitative PCR detection primer set and probe set and reagent kit and preparation method thereof |
| CN105713993A (en) * | 2016-02-26 | 2016-06-29 | 深圳市亿立方生物技术有限公司 | PCR primer set for detecting multiple respiratory viruses, probe set and kit |
| CN108239676A (en) * | 2016-12-23 | 2018-07-03 | 上海星耀医学科技发展有限公司 | A kind of parainfluenza virus one-step method fluorescence parting RT-PCR detection kit |
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| CN103114154A (en) * | 2013-02-25 | 2013-05-22 | 湖北朗德医疗科技有限公司 | Rhinovirus real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) detection kit and application thereof |
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| CN104059995A (en) * | 2014-06-06 | 2014-09-24 | 深圳市疾病预防控制中心 | Kit and method for simultaneously detecting respiratory syncytial virus, adenovirus and human bocavirus |
| CN104846121A (en) * | 2015-04-18 | 2015-08-19 | 湖北创瑞生物科技有限公司 | Virus triple fluorescence quantitative RT-PCR detection method and virus triple fluorescence quantitative RT-PCR detection kit |
| CN105543414A (en) * | 2016-01-22 | 2016-05-04 | 广州医科大学附属第一医院 | Respiratory syncytial virus A/B subtype multiplex fluorescence quantitative PCR detection primer set and probe set and reagent kit and preparation method thereof |
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| CN105713993A (en) * | 2016-02-26 | 2016-06-29 | 深圳市亿立方生物技术有限公司 | PCR primer set for detecting multiple respiratory viruses, probe set and kit |
| CN108239676A (en) * | 2016-12-23 | 2018-07-03 | 上海星耀医学科技发展有限公司 | A kind of parainfluenza virus one-step method fluorescence parting RT-PCR detection kit |
| CN113186342A (en) * | 2021-04-07 | 2021-07-30 | 吉林双正医疗科技有限公司 | 18 unite respiratory track virus nucleic acid and unite detection device |
| CN113186342B (en) * | 2021-04-07 | 2023-03-14 | 吉林双正医疗科技有限公司 | 18 ally oneself with respiratory virus nucleic acid and unite detection device |
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