CN105018648A - Kit for detecting respiratory viruses and application thereof - Google Patents
Kit for detecting respiratory viruses and application thereof Download PDFInfo
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- CN105018648A CN105018648A CN201510468571.2A CN201510468571A CN105018648A CN 105018648 A CN105018648 A CN 105018648A CN 201510468571 A CN201510468571 A CN 201510468571A CN 105018648 A CN105018648 A CN 105018648A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/702—Specific hybridization probes for retroviruses
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention discloses a kit for detecting respiratory viruses and an application thereof. The kit comprises a primer pair group for detecting the respiratory viruses, wherein the primer pair group consists of 16 primer pairs, the sequences of which are shown in sequences 1 to 32 in a sequence table. The kit provided by the invention supports high throughput, can be used for rapidly and accurately detecting the infection of conventional respiratory viruses, and can clinically acquire a detection result of 16 virus indexes in one hour, which is not only faster than the real-time fluorescence quantitative PCR method generally used at present, but also has an important significance for rapidly assisting and instructing the treatment and medicine application. Meanwhile, the multi-index detection can also be used for investigating regional epidemiology and monitoring the epidemic situation so as to research the prevalence state of respiratory virus infection in China.
Description
Technical field
The invention belongs to nucleic acid amplification technologies field, relating to a kind of test kit for detecting Respirovirus and application thereof.
Background technology
Respiratory tract infection is divided into upper respiratory tract infection and lower respiratory infection, main pathogens comprises bacterium, virus, mycoplasma, chlamydozoan, fungi etc., wherein main based on viral infection, particularly upper breathing in respiratory tract infection has more than 80% to be viral.These disease four seasons, any age all can fall ill, and by containing the virulent spittle, droplet, or the apparatus through polluting is propagated.Normal when Abwehrkraft des Koepers reduces, as caught cold, tired, the situation such as to drench with rain, the former virus that existed or invaded by the external world or bacterium, ramp is bred, and causes infection, recovery from illness in general 5-7 days of being grown up.
Childhood infection respiratory pathogen brings out acute respiratory infection (ARI) usually, ARI is infant, children's common disease, Chang Jifa bronchitis, pneumonia, nasal sinusitis, minority can complicated with acute meningitis, myocarditis, ephritis, rheumatic fever etc., and complication serious harm children's is healthy.Confirmed that the ARI of 95% is caused by the pathogenic agent beyond bacterium, virus is ARI main pathogens.Children in different ages is distinguished to some extent to virus susceptibility, and according to WHO data presentation, respiratory tract infection is the dead the first reason of underage child in world wide (<5 year), can cause about 5,000,000 death of child every year.The ill number of times average out to 3-8 times/year of children with acute respiratory tract infection, the ratio about 1 ~ 3% infected in children at 1 years old, school-ager to 5% ~ 10%.The viral species popular due to each period and type are not quite similar, and a few different virus again can polyinfection sometimes, therefore has very important meaning to the monitoring of childrens respiratory tract virus causing disease.Rapid&Early diagnosis respiratory virus infection can in time guiding clinical treatment for choose reasonable antiviral antibacterial medicine provide according to and prevent in abuse of antibiotics significant.
Respirovirus refers to the virus that can infect respiratory tract or be undertaken by respiratory tract propagating, and at least relates to 8 sections, other virus of 200 multiple-types.The virus of clinical common caused respiratory tract infection comprises influenza A virus (InfA) and the Influenza B virus (InfB) of orthomyxoviridae family, the parainfluenza virus 1-3 type (PIV1-PIV3) of Paramyxoviridae, respiratory syncytial virus (RSV), human metapneumovirus (hMPV), the adenovirus (AdV) of Adenoviridae, the rhinovirus (HRV) of Pironavirus section, coronavirus (Corn) and enterovirus (Entv) etc.The method of the common Respirovirus of current detection is more, but all Shortcomings, as Electronic Speculum detection method complex and expensive, Viral isolation is consuming time extremely long and false negative is more, after taking cultivation results, usually lose clinical meaning, what euzymelinked immunosorbent assay (ELISA) detected is special viral antibody but once only can detects a kind of virus.And based on the detection of nucleic acids of nucleic acid amplification, due to speed fast (can complete detection in usual 3 hours), highly sensitive, specificity good, becomes gold standard just gradually in field of virus detection.
Rely on the amplification technique (Nucleic Acid Sequence Based Amplification) of nucleotide sequence, i.e. NASBA amplification technique, is mediated by pair of primers, specificity the homogeneous continuously process of single stranded RNA being carried out to constant-temperature amplification in vitro.Under 41 DEG C of constant temperatures, in speed of response, NASBA increases 1h can by template ribonucleic acid amplification about 10
9~ 10
12doubly, common PCR reaction needed 2 ~ 3h.In detection sensitivity, NASBA can detect the trace target lower than 10genecopies/ μ l in solution, and the detectability of PCR is at 100gene copies/ μ about l.Therefore, NASBA technology is that amplification efficiency or detection sensitivity are all higher than RT-PCR technology.And only needing the constant temperature of 41 DEG C due to the reaction of NASBA, water-bath can meet reaction requirement, and do not need the temperature control device that the Standard PCR such as complicated heating and cooling rely on, therefore the instrument cost of NASBA technology is very cheap.Be combined with corresponding detection technique, NASBA also has the plurality of advantages such as easy and simple to handle, high specificity.
Summary of the invention
First object of the present invention is to provide a kind of primer pair group for detecting Respirovirus.
Primer pair group for detecting Respirovirus provided by the present invention, is made up of following 16 primer pairs:
The primer pair 1 be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2; The primer pair 2 be made up of two single strand dnas shown in sequence in sequence table 3 and sequence 4; The primer pair 3 be made up of two single strand dnas shown in sequence in sequence table 5 and sequence 6; The primer pair 4 be made up of two single strand dnas shown in sequence in sequence table 7 and sequence 8; The primer pair 5 be made up of two single strand dnas shown in sequence in sequence table 9 and sequence 10; The primer pair 6 be made up of two single strand dnas shown in sequence in sequence table 11 and sequence 12; The primer pair 7 be made up of two single strand dnas shown in sequence in sequence table 13 and sequence 14; The primer pair 8 be made up of two single strand dnas shown in sequence in sequence table 15 and sequence 16; The primer pair 9 be made up of two single strand dnas shown in sequence in sequence table 17 and sequence 18; The primer pair 10 be made up of two single strand dnas shown in sequence in sequence table 19 and sequence 20; The primer pair 11 be made up of two single strand dnas shown in sequence in sequence table 21 and sequence 22; The primer pair 12 be made up of two single strand dnas shown in sequence in sequence table 23 and sequence 24; The primer pair 13 be made up of two single strand dnas shown in sequence in sequence table 25 and sequence 26; The primer pair 14 be made up of two single strand dnas shown in sequence in sequence table 27 and sequence 28; The primer pair 15 be made up of two single strand dnas shown in sequence in sequence table 29 and sequence 30; The primer pair 16 be made up of two single strand dnas shown in sequence in sequence table 31 and sequence 32.
Wherein, described primer pair 1 (InfA_TY) is for the influenza A virus that increases; Described primer pair 2 (InfA_H1) is for the influenza A virus H1 hypotype that increases; Described primer pair 3 (InfA_H3) is for the influenza A virus H3 hypotype that increases; Described primer pair 4 (InfB_TY) is for the Influenza B virus that increases; Described primer pair 5 (RSV_TY) is for the respiratory syncytial virus that increases; Described primer pair 6 (HRV_TY) is for rhinovirus of increasing; Described primer pair 7 (ADV_TY) is for the adenovirus that increases; Described primer pair 8 (PIV1) is for the parainfluenza virus I type that increases; Described primer pair 9 (PIV2) is for the parainfluenza virus II type that increases; Described primer pair 10 (PIV3) is for the parainfluenza virus type III that increases; Described primer pair 11 (Corn_OC) is for the HCoV-HKU1/OC43 that increases; Described primer pair 12 (Corn_NL) is for the HCoV-229E/NL63 that increases; Described primer pair 13 (hMPV_TY) is for the human metapneumovirus that increases; Described primer pair 14 (Entv_TY) is for enterovirus of increasing; Described primer pair 15 (EV71_VP1) is for the enterovirus EV 71 type that increases; Described primer pair 16 (CA16_VP1) is for the enterovirus CA16 type that increases.
Second object of the present invention is to provide a kind of complete single stranded DNA for detecting Respirovirus.
Complete single stranded DNA for detecting Respirovirus provided by the present invention, is made up of probe groups and described primer pair group; Described probe groups is made up of following 16 ssDNA probe: the ssDNA probe 1 in sequence table shown in sequence 33; SsDNA probe 2 in sequence table shown in sequence 34; SsDNA probe 3 in sequence table shown in sequence 35; SsDNA probe 4 in sequence table shown in sequence 36; SsDNA probe 5 in sequence table shown in sequence 37; SsDNA probe 6 in sequence table shown in sequence 38; SsDNA probe 7 in sequence table shown in sequence 39; SsDNA probe 8 in sequence table shown in sequence 40; SsDNA probe 9 in sequence table shown in sequence 41; SsDNA probe 10 in sequence table shown in sequence 42; SsDNA probe 11 in sequence table shown in sequence 43; SsDNA probe 12 in sequence table shown in sequence 44; SsDNA probe 13 in sequence table shown in sequence 45; SsDNA probe 14 in sequence table shown in sequence 46; SsDNA probe 15 in sequence table shown in sequence 47; SsDNA probe 16 in sequence table shown in sequence 48.
Wherein, described ssDNA probe 1 (InfA_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 1; Described ssDNA probe 2 (InfA_H1_MB) is for the amplification of primer pair described in Real-Time Monitoring 2; Described ssDNA probe 3 (InfA_H3_MB) is for the amplification of primer pair described in Real-Time Monitoring 3; Described ssDNA probe 4 (InfB_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 4; Described ssDNA probe 5 (RSV_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 5; Described ssDNA probe 6 (HRV_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 6; Described ssDNA probe 7 (ADV_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 7; Described ssDNA probe 8 (PIV1_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 8; Described ssDNA probe 9 (PIV2_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 9; Described ssDNA probe 10 (PIV3_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 10; Described ssDNA probe 11 (Corn_OC_MB) is for the amplification of primer pair described in Real-Time Monitoring 11; Described ssDNA probe 12 (Corn_NL_MB) is for the amplification of primer pair described in Real-Time Monitoring 12; Described ssDNA probe 13 (hMPV_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 13; Described ssDNA probe 14 (Entv_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 14; Described ssDNA probe 15 (EV71_VP1_MB) is for the amplification of primer pair described in Real-Time Monitoring 15; Described ssDNA probe 16 (CA16_VP1_MB) is for the amplification of primer pair described in Real-Time Monitoring 16.
5 ' end of the every bar ssDNA probe in described probe groups is all marked with fluorescent reporter group FAM, and 3 ' end is all marked with fluorescent quenching group TAMRA.
In described complete single stranded DNA provided by the present invention, the ssDNA probe of each primer pair and correspondence thereof can be packaged in separately in a packaging.As described in primer pair 1 and as described in ssDNA probe 1 be packaged in first packaging in; Described primer pair 2 and described ssDNA probe 2 are packaged in second packaging; Described primer pair 3 and described ssDNA probe 3 are packaged in the 3rd packaging; Described primer pair 4 and described ssDNA probe 4 are packaged in the 4th packaging; Described primer pair 5 and described ssDNA probe 5 are packaged in the 5th packaging; Described primer pair 6 and described ssDNA probe 6 are packaged in the 6th packaging; Described primer pair 7 and described ssDNA probe 7 are packaged in the 7th packaging; Described primer pair 8 and described ssDNA probe 8 are packaged in the 8th packaging; Described primer pair 9 and described ssDNA probe 9 are packaged in the 9th packaging; Described primer pair 10 and described ssDNA probe 10 are packaged in the tenth packaging; Described primer pair 11 and described ssDNA probe 1 are packaged in the 11 packaging; Described primer pair 12 and described ssDNA probe 12 are packaged in the 12 packaging; Described primer pair 13 and described ssDNA probe 13 are packaged in the 13 packaging; Described primer pair 14 and described ssDNA probe 14 are packaged in the 14 packaging; Described primer pair 15 and described ssDNA probe 15 are packaged in the 15 packaging; Described primer pair 16 and described ssDNA probe 16 are packaged in the 16 packaging.
3rd object of the present invention is to provide a kind of test kit for detecting Respirovirus.
Test kit for detecting Respirovirus provided by the present invention, containing described primer pair group or described complete single stranded DNA.
Described test kit also can contain internal reference primer pair and internal reference probe.Described internal reference primer pair is the primer pair GAPD_IC for house-keeping gene GAPDH mRNA in the human genome that increases, and is made up of two single strand dnas shown in sequence in sequence table 49 and sequence 50.Described internal reference probe (GAPD_IC_MB) is the ssDNA probe shown in sequence in sequence table 51, for the amplification of internal reference primer pair (GAPD_IC) described in Real-Time Monitoring.
5 ' end of described internal reference probe is marked with fluorescent reporter group FAM, and 3 ' end is marked with fluorescent quenching group TAMRA.
Also constant-temperature amplification damping fluid and constant-temperature amplification enzyme solution can be contained in described test kit.
The solvent of described constant-temperature amplification damping fluid is water, solute and concentration as follows: the Tris-HCL of 200mM pH 8.0,50mMDTT, 10mM dNTP, 10mM rNTP, 80mM MgCl
2, 450mM KCl, 15% volumn concentration DMSO, 1M sorbyl alcohol, 20mM tetramethyl ammonium chloride.
The solvent of described constant-temperature amplification enzyme solution is water, solute and concentration as follows: AMV reversed transcriptive enzyme 1U/ μ l, t7 rna polymerase 5U/ μ l, ribonuclease H 0.5U/ μ l, Pyrophosphate phosphohydrolase 0.5U/ μ l, RNA enzyme inhibitors 5U/ μ l, BSA 0.5 μ g/ μ l.
Also dish-style chip can be contained, as 24 reaction chamber dish-style chips in described test kit.
In the present invention, described 24 reaction chamber dish-style chips are the supporting dish-style chip of " the brilliant core RTisochipTM-A constant-temperature amplification micro-fluidic chip foranalysis of nucleic acids instrument " that Capitalbio Corporation Co., Ltd. produces, and its model is 1 × 24.
1# to the 18# reaction chamber of described dish-style chip deposits dry following (1)-(18) respectively:
(1) described primer pair 1 and described ssDNA probe 1; (2) described primer pair 2 and described ssDNA probe 2; (3) described primer pair 3 and described ssDNA probe 3; (4) described primer pair 4 and described ssDNA probe 4; (5) described primer pair 5 and described ssDNA probe 5; (6) described primer pair 6 and described ssDNA probe 6; (7) described primer pair 7 and described ssDNA probe 7; (8) described primer pair 8 and described ssDNA probe 8; (9) described primer pair 9 and described ssDNA probe 9; (10) described primer pair 10 and described ssDNA probe 10; (11) described primer pair 11 and described ssDNA probe 11; (12) described primer pair 12 and described ssDNA probe 12; (13) described primer pair 13 and described ssDNA probe 13; (14) described primer pair 14 and described ssDNA probe 14; (15) described primer pair 15 and described ssDNA probe 15; (16) described primer pair 16 and described ssDNA probe 16; (17) described internal reference primer pair and described internal reference probe; (18) negative controls.
Described negative controls specifically can be the water (Rnase-free water) without RNA enzyme.
Wherein, method primer and probe being embedded into dish-style chip is: primer, the probe corresponding with described primer, agarose are mixed, be mixed with mixing solutions, make described primer, described probe and the final concentration of described agarose in described mixing solutions be respectively 0.2 μM, 50nM, 0.1% (mass percentage); Get mixing solutions described in 1 μ l and click and enter corresponding dish-style chip reaction chamber, after drying in clean super clean bench, compressing tablet encapsulates, and makes after punching press vacuumizes.
Described primer pair group or complete single stranded DNA or test kit are detecting or non-diagnostic object application in auxiliary detection Respirovirus also belongs to protection scope of the present invention.
In the application, by constant-temperature amplification enzyme solution described in constant-temperature amplification damping fluid described in 15 μ l and 10 μ l, be injected in described dish-style chip after mixing with 25 μ l sample to be tested solution, isothermal reaction 1h at 41 DEG C.
In the application, can Nasopharyngeal swabs as sample to be tested; Accordingly, described sample to be tested solution can be the RNA extracted from Nasopharyngeal swabs.
In the present invention, described Respirovirus specifically can be at least one as follows: influenza A virus, influenza A virus H1 hypotype, influenza A virus H3 hypotype, Influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, parainfluenza virus I type, parainfluenza virus II type, parainfluenza virus type III, HCoV-HKU1/OC43, HCoV-229E/NL63, human metapneumovirus, enterovirus, enterovirus EV 71 type and enterovirus CA16 type.
Test kit provided by the invention supports high-throughput, common respiratory virus infection can be detected quickly and accurately, for clinical, the detected result that can obtain 16 kinds of viral indexs in 1 hour not only faster than the real time fluorescence quantifying PCR method comparatively generally adopted at present, and for quick auxiliary direction treatment and medication also significant.Meanwhile, the detection of multi objective also may be used for regional epidemiological survey and epidemic situation monitoring, to study the popularity of respiratory virus infection in China.
Accompanying drawing explanation
Fig. 1 is dish-style chip and primer sample application cavity schematic diagram.
Fig. 2 is 16 kinds of Respirovirus reference material dish-style chip detection result figure.Wherein, A is the detected result of the recombinant plasmid pUC19-InfA containing influenza A virus MP target gene at 1# reaction chamber; B is the detected result of the recombinant plasmid pUC19-InfA_H1 containing influenza A virus H1 hypotype target gene at 2# reaction chamber; C is the detected result of the recombinant plasmid pUC19-InfA_H3 containing influenza A virus H3 hypotype target gene at 3# reaction chamber; D is the detected result of the recombinant plasmid pUC19-InfB containing Influenza B virus MP target gene at 4# reaction chamber; E is the detected result of the recombinant plasmid pUC19-RSV containing respiratory syncytial virus target gene at 5# reaction chamber; F is the detected result of the recombinant plasmid pUC19-HRV containing rhinovirus target gene at 6# reaction chamber; G is the detected result of the recombinant plasmid pUC19-ADV containing adenovirus target gene at 7# reaction chamber; H is the detected result of the recombinant plasmid pUC19-PIV1 containing parainfluenza virus I type target gene at 8# reaction chamber; I is the detected result of the recombinant plasmid pUC19-PIV2 containing parainfluenza virus II type target gene at 9# reaction chamber; J is the detected result of the recombinant plasmid pUC19-PIV3 containing parainfluenza virus type III target gene at 10# reaction chamber; K is for the recombinant plasmid pUC19-Corn_OC containing HCoV-HKU1/OC43 target gene is in the detected result of 11# reaction chamber; L is for the recombinant plasmid pUC19-Corn_NL containing HCoV-229E/NL63 target gene is in the detected result of 12# reaction chamber; M is the detected result of the recombinant plasmid pUC19-hMPV containing human metapneumovirus target gene at 13# reaction chamber; N is the detected result of the recombinant plasmid pUC19-Entv containing enterovirus target gene at 14# reaction chamber; O is the detected result of the recombinant plasmid pUC19-EV71 containing enterovirus EV 71 target gene at 15# reaction chamber; P is the detected result of the recombinant plasmid pUC19-CA16 containing enterovirus CA16 target gene at 16# reaction chamber.In A-P, E1 represents that template is 10
1copy/μ l, by that analogy, E5 represents that template is 10
5copy/μ l; NC represents negative control.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1, for detecting preparation and the use thereof of the test kit of Respirovirus
One, for detecting the preparation of the test kit of Respirovirus
Test kit for detecting Respirovirus provided by the present invention is composed as follows:
1, constant-temperature amplification damping fluid
The solvent of constant-temperature amplification damping fluid is water, solute and concentration as follows: 200mM Tris-HCL (pH 8.0), 50mM DTT, 10mM dNTP, 10mM rNTP, 80mM MgCl
2, 450mM KCl, 15% volumn concentration DMSO, 1M sorbyl alcohol, 20mM tetramethyl ammonium chloride.
2, constant-temperature amplification enzyme solution
The solvent of constant-temperature amplification enzyme solution is water, solute and concentration as follows: AMV reversed transcriptive enzyme 1U/ μ l, t7 rna polymerase 5U/ μ l, ribonuclease H 0.5U/ μ l, Pyrophosphate phosphohydrolase 0.5U/ μ l, RNA enzyme inhibitors 5U/ μ l, BSA 0.5 μ g/ μ l.
3,24 reaction chamber dish-style chips of primer pair and ssDNA probe are mounted with
Described 24 reaction chamber dish-style chips are the supporting dish-style chip of " the brilliant core RTisochipTM-A constant-temperature amplification micro-fluidic chip foranalysis of nucleic acids instrument " that Capitalbio Corporation Co., Ltd. produces, and its model is 1 × 24, and schematic diagram as shown in Figure 1.
1# to the 18# reaction chamber of described 24 reaction chamber dish-style chips deposits dry following (1)-(18) respectively:
(1) primer pair 1 and ssDNA probe 1; (2) primer pair 2 and ssDNA probe 2; (3) primer pair 3 and ssDNA probe 3; (4) primer pair 4 and described DNA probe 4; (5) primer pair 5 and ssDNA probe 5; (6) primer pair 6 and ssDNA probe 6; (7) primer pair 7 and ssDNA probe 7; (8) primer pair 8 and ssDNA probe 8; (9) primer pair 9 and ssDNA probe 9; (10) primer pair 10 and ssDNA probe 10; (11) primer pair 11 and ssDNA probe 11; (12) primer pair 12 and ssDNA probe 12; (13) primer pair 13 and ssDNA probe 13; (14) primer pair 14 and ssDNA probe 14; (15) primer pair 15 and ssDNA probe 15; (16) primer pair 16 and ssDNA probe 16; (17) internal reference primer pair and internal reference probe; (18) negative controls.Described negative controls is specially the water (Rnase-free water) without RNA enzyme.
Wherein, described primer pair 1 (InfA_TY), for the influenza A virus that increases, is made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2; Described primer pair 2 (InfA_H1), for the influenza A virus H1 hypotype that increases, is made up of two single strand dnas shown in sequence in sequence table 3 and sequence 4; Described primer pair 3 (InfA_H3), for the influenza A virus H3 hypotype that increases, is made up of two single strand dnas shown in sequence in sequence table 5 and sequence 6; Described primer pair 4 (InfB_TY), for the Influenza B virus that increases, is made up of two single strand dnas shown in sequence in sequence table 7 and sequence 8; Described primer pair 5 (RSV_TY), for the respiratory syncytial virus that increases, is made up of two single strand dnas shown in sequence in sequence table 9 and sequence 10; Described primer pair 6 (HRV_TY), for rhinovirus of increasing, is made up of two single strand dnas shown in sequence in sequence table 11 and sequence 12; Described primer pair 7 (ADV_TY), for the adenovirus that increases, is made up of two single strand dnas shown in sequence in sequence table 13 and sequence 14; Described primer pair 8 (PIV1), for the parainfluenza virus I type that increases, is made up of two single strand dnas shown in sequence in sequence table 15 and sequence 16; Described primer pair 9 (PIV2), for the parainfluenza virus II type that increases, is made up of two single strand dnas shown in sequence in sequence table 17 and sequence 18; Described primer pair 10 (PIV3), for the parainfluenza virus type III that increases, is made up of two single strand dnas shown in sequence in sequence table 19 and sequence 20; Described primer pair 11 (Corn_OC), for the HCoV-HKU1/OC43 that increases, is made up of two single strand dnas shown in sequence in sequence table 21 and sequence 22; Described primer pair 12 (Corn_NL), for the HCoV-229E/NL63 that increases, is made up of two single strand dnas shown in sequence in sequence table 23 and sequence 24; Described primer pair 13 (hMPV_TY), for the human metapneumovirus that increases, is made up of two single strand dnas shown in sequence in sequence table 25 and sequence 26; Described primer pair 14 (Entv_TY), for enterovirus of increasing, is made up of two single strand dnas shown in sequence in sequence table 27 and sequence 28; Described primer pair 15 (EV71_VP1), for the enterovirus EV 71 type that increases, is made up of two single strand dnas shown in sequence in sequence table 29 and sequence 30; Described primer pair 16 (CA16_VP1), for the enterovirus CA16 type that increases, is made up of two single strand dnas shown in sequence in sequence table 31 and sequence 32.Described internal reference primer pair (GAPD_IC), for house-keeping gene GAPDH mRNA in the human genome that increases, is made up of two single strand dnas shown in sequence in sequence table 49 and sequence 50.
Described ssDNA probe 1 (InfA_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 1, and its nucleotides sequence is classified as sequence 33 in sequence table; Described ssDNA probe 2 (InfA_H1_MB) is for the amplification of primer pair described in Real-Time Monitoring 2, and its nucleotides sequence is classified as sequence 34 in sequence table; Described ssDNA probe 3 (InfA_H3_MB) is for the amplification of primer pair described in Real-Time Monitoring 3, and its nucleotides sequence is classified as sequence 35 in sequence table; Described ssDNA probe 4 (InfB_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 4, and its nucleotides sequence is classified as sequence 36 in sequence table; Described ssDNA probe 5 (RSV_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 5, and its nucleotides sequence is classified as sequence 37 in sequence table; Described ssDNA probe 6 (HRV_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 6, and its nucleotides sequence is classified as sequence 38 in sequence table; Described ssDNA probe 7 (ADV_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 7, and its nucleotides sequence is classified as sequence 39 in sequence table; Described ssDNA probe 8 (PIV1_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 8, and its nucleotides sequence is classified as sequence 40 in sequence table; Described ssDNA probe 9 (PIV2_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 9, and its nucleotides sequence is classified as sequence 41 in sequence table; Described ssDNA probe 10 (PIV3_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 10, and its nucleotides sequence is classified as sequence 42 in sequence table; Described ssDNA probe 11 (Corn_OC_MB) is for the amplification of primer pair described in Real-Time Monitoring 11, and its nucleotides sequence is classified as sequence 43 in sequence table; Described ssDNA probe 12 (Corn_NL_MB) is for the amplification of primer pair described in Real-Time Monitoring 12, and its nucleotides sequence is classified as sequence 44 in sequence table; Described ssDNA probe 13 (hMPV_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 13, and its nucleotides sequence is classified as sequence 45 in sequence table; Described ssDNA probe 14 (Entv_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 14, and its nucleotides sequence is classified as sequence 46 in sequence table; Described ssDNA probe 15 (EV71_VP1_MB) is for the amplification of primer pair described in Real-Time Monitoring 15, and its nucleotides sequence is classified as sequence 47 in sequence table; Described ssDNA probe 16 (CA16_VP1_MB) is for the amplification of primer pair described in Real-Time Monitoring 16, and its nucleotides sequence is classified as sequence 48 in sequence table.Described internal reference probe (GAPD_IC_MB) is for the amplification of internal reference primer pair (GAPD_IC) described in Real-Time Monitoring, and its nucleotides sequence is classified as sequence 51 in sequence table.5 ' end of ssDNA probe described in every bar and described internal reference probe (GAPD_IC_MB) is all marked with fluorescent reporter group FAM, and 3 ' end is all marked with fluorescent quenching group TAMRA.
Wherein, method primer and probe being embedded into dish-style chip is: by primer, corresponding probe, agarose mixing, the final concentration being mixed with three kinds of compositions be followed successively by 0.2 μM, 50nM, 0.1% (mass percentage) mixing solutions, get 1 this solution of μ l and click and enter corresponding dish-style chip reaction chamber, after drying in clean super clean bench, compressing tablet encapsulates, and makes after punching press vacuumizes.
Two, for detecting the using method of the test kit of Respirovirus
1, reaction system preparation
Get 15 μ l constant-temperature amplification damping fluids (see step one 1), 10 μ l constant-temperature amplification enzyme solution (see step one 2), 25 μ l sample to be tested liquid are mixed into 50 μ l reaction solns, the 24 reaction chamber dish-style chips (see step one 3) being mounted with primer pair and ssDNA probe are injected, Quick spin 30s after sealing membrana oralis after vortex concussion evenly.
2, isothermal amplification reactions and detection
Dish-style chip is placed in rich Isochip-RT constant-temperature amplification instrument difficult to understand, 41 DEG C of reactions 1 hour is set, and complete real-time fluorescence scanning simultaneously.
3, result judges
After reaction terminates, the genome whether containing corresponding Respirovirus in described sample to be tested liquid is determined as follows: if described testing sample produces S type amplification curve, then the genome containing corresponding Respirovirus in described sample to be tested liquid according to the amplification curve of sample in each reaction chamber; Otherwise, then the genome not containing corresponding Respirovirus in described sample to be tested liquid.
Embodiment 2, for detecting sensitivity and the specificity analyses of the test kit of Respirovirus
One, the preparation of reference material RNA nucleic acid
1, the plasmid including viral targets gene is built
(1) plasmid containing influenza A virus NP target gene
By 1 ~ 1541 section (the Genbank Sequence ID:gb|KM366530.1| of influenza A virus NP target gene sequence of influenza A virus NP target gene sequence, Update Date:2014-9-27) be inserted into pUC19 carrier (sky with biochemical corp's product) multiple clone site EcoR V between, obtain recombinant plasmid pUC19-InfA.
(2) plasmid containing influenza A virus H1 hypotype target gene
By 1 ~ 1698 section (the Genbank Sequence ID:gb|GQ475727.1| of influenza A virus H1 hypotype target gene sequence of influenza A virus H1 hypotype target gene sequence, Update Date:2009-8-19) be inserted between pUC19 carrier (sky is with biochemical corp's product) EcoR V, obtain recombinant plasmid pUC19-InfA_H1.
(3) plasmid containing influenza A virus H3 hypotype target gene
By 1 ~ 1701 section (the Genbank Sequence ID:gb|KJ567658.1| of influenza A virus H3 hypotype target gene sequence of influenza A virus H3 hypotype target gene sequence, Update Date:2014-4-6) be inserted into pUC19 carrier (sky with biochemical corp's product) multiple clone site EcoR V between, obtain recombinant plasmid pUC19-InfA_H3.
(4) plasmid containing Influenza B virus MP target gene
By 2 ~ 1100 sections (the Genbank Sequence ID:gb|EU305617.1| of Influenza B virus MP target gene sequence of Influenza B virus MP target gene sequence, Update Date:2007-12-5) be inserted into pUC19 carrier (sky with biochemical corp's product) multiple clone site EcoR V between, obtain recombinant plasmid pUC19-InfB.
(5) plasmid containing respiratory syncytial virus target gene
By 5680 ~ 7390 sections (the Genbank Sequence ID:gb|KJ627648.1| of respiratory syncytial virus target gene sequence of respiratory syncytial virus target gene sequence, Update Date:2014-4-16) be inserted into pUC19 carrier (sky with biochemical corp's product) multiple clone site EcoR V between, obtain recombinant plasmid pUC19-RSV.
(6) plasmid containing rhinovirus target gene
By 1 ~ 1175 section (the Genbank Sequence ID:gb|M16248.1| of rhinovirus target gene sequence of rhinovirus target gene sequence, Update Date:2003-2-7) be inserted into pUC19 carrier (sky with biochemical corp's product) multiple clone site EcoR V between, obtain recombinant plasmid pUC19-HRV.
(7) plasmid containing adenovirus target gene
By 20035-22649 section (the Genbank SequenceID:gb|KF268314.1| of adenovirus target gene sequence of adenovirus target gene sequence, Update Date:2013-9-12) be inserted into pUC19 carrier (sky with biochemical corp's product) multiple clone site EcoR V between, obtain recombinant plasmid pUC19-ADV.
(8) plasmid containing parainfluenza virus I type target gene
Between the multiple clone site EcoR V 464 ~ 1296 sections (the Genbank Sequence ID:gb|JF416791.1| of parainfluenza virus I type target gene sequence, Update Date:2014-12-31) of parainfluenza virus I type target gene sequence being inserted into pUC19 carrier (sky with biochemical corp's product).Obtain recombinant plasmid pUC19-PIV1.
(9) plasmid containing parainfluenza virus II type target gene
Between the multiple clone site EcoR V 2568 ~ 3629 sections (the Genbank Sequence ID:gb|DQ072586.1| of parainfluenza virus II type target gene sequence, Update Date:2008-9-4) of parainfluenza virus II type target gene sequence being inserted into pUC19 carrier (sky with biochemical corp's product).Obtain recombinant plasmid pUC19-PIV2.
(10) plasmid containing parainfluenza virus type III target gene
Between the multiple clone site EcoR V 211 ~ 1190 sections (the Genbank Sequence ID:dbj|AB623488.1| of parainfluenza virus type III target gene sequence, Update Date:2014-7-26) of parainfluenza virus type III target gene sequence being inserted into pUC19 carrier (sky with biochemical corp's product).Obtain recombinant plasmid pUC19-PIV3.
(11) plasmid containing HCoV-HKU1/OC43 target gene
By 20438 ~ 21549 sections (the Genbank Sequence ID:gb|KF686344.1| of HCoV-HKU1/OC43 target gene sequence of HCoV-HKU1/OC43 target gene sequence, Update Date:2014-9-26) be inserted into pUC19 carrier (sky with biochemical corp's product) multiple clone site EcoR V between, obtain recombinant plasmid pUC19-Corn_OC.
(12) plasmid containing HCoV-229E/NL63 target gene
By 15715 ~ 17379 sections (the Genbank Sequence ID:gb|JQ765566.1| of HCoV-229E/NL63 target gene sequence of HCoV-229E/NL63 target gene sequence, Update Date:2014-9-26) be inserted into pUC19 carrier (sky with biochemical corp's product) multiple clone site EcoR V between, obtain recombinant plasmid pUC19-Corn_NL.
(13) plasmid containing human metapneumovirus target gene
By 3513 ~ 4544 sections (the Genbank Sequence ID:gb|KC562240.1| of human metapneumovirus target gene sequence of human metapneumovirus target gene sequence, Update Date:2013-4-21) be inserted into pUC19 carrier (sky with biochemical corp's product) multiple clone site EcoR V between, obtain recombinant plasmid pUC19-hMPV.
(14) plasmid containing enterovirus target gene
By 5 ~ 1705 sections (the Genbank Sequence ID:gb|KC755230.1| of enterovirus Entv target gene sequence of enterovirus Entv target gene sequence, Update Date:2013-12-17) be inserted into pUC19 carrier (sky with biochemical corp's product) multiple clone site EcoR V between, obtain recombinant plasmid pUC19-Entv.
(15) plasmid containing enterovirus EV 71 target gene
By 4 ~ 1695 sections (the Genbank Sequence ID:gb|HM245928.1| of enterovirus EV 71 target gene sequence of enterovirus EV 71 target gene sequence, Update Date:2011-9-27) be inserted into pUC19 carrier (sky with biochemical corp's product) multiple clone site EcoR V between, obtain recombinant plasmid pUC19-EV71.
(16) plasmid containing enterovirus CA16 target gene
By 1392 ~ 2564 sections (the Genbank Sequence ID:gb|AY790926.1| of enterovirus CA16 target gene sequence of enterovirus CA16 target gene sequence, Update Date:2005-8-9) be inserted into pUC19 carrier (sky with biochemical corp's product) multiple clone site EcoR V between, obtain recombinant plasmid pUC19-CA16.
2, the preparation of reference material RNA nucleic acid
For examination recombinant plasmid: 16 kinds of step 1 structure contain the recombinant plasmid of corresponding viral targets gene.
(1) enzyme is cut: first cut 2h by each for examination recombinant plasmid EcoRI restriction endonuclease 37 DEG C of enzymes.
(2) transcribe: by 5 μ l 5 × Transcription Optimized Buffer (Promega), 2U t7 rna polymerase, 10mMDTT (Promega), 10U recombinant RNA enzyme inhibitors (Promega), 2mM rNTP, it is the reaction system of 50 μ l that digestion products 5 μ l prepares cumulative volume, vibrations evenly latter 37 DEG C transcribe 4h.
(3) digest: the DNA digestive ferment (rDnaseI, the 5U/ μ l) system after having transcribed being added 1 μ l, shake centrifugal, hatch 20min for 37 DEG C.
(4) purifying: use RNA purification kit product (its catalog number is 740948) the purifying transcription product RNA:a as follows of Macherey-Nagel company) prepare RA1-C
2h
5oH mixed solution: with RA1:C
2h
5the proportions of OH=1:1 (volume ratio).In the transcription product of every 100 μ l, need to add 600 μ l RA1-C
2h
5oH mixed solution, i.e. 300 μ lRA1+300 μ l C
2h
5oH solution, here need according to the pipe number of transcription product calculate join the volume of mixed solution.If product is less than 100 μ l, then the amount of product is mended to 100 μ l with water and (in every pipe product, namely add the sky root Rnase-free H of 50 μ l
2o).B) ready RA1-C is before incited somebody to action
2h
5oH mixed solution is assigned in 1.5ml centrifuge tube, two pipes, and 100 μ l products are transferred in corresponding centrifuge tube by often pipe 600 μ l, totally 700 μ l in pipe, and fully concussion is centrifugal.C) prepare two adsorption columns, and carry out corresponding mark, be transferred to by 700 μ l product mixture (adsorption column maximum capacity is 700 μ l) in adsorption column, the centrifugal 2min of 1.2 ten thousand rpm, outwells lower floor's solution.D) in adsorption column, add the RA3 of 700 μ l, place 1min, make it can be dispersed in the bottom of adsorption column fully, the centrifugal 2min of 1.2 ten thousand rpm, outwells lower floor's solution.E) in adsorption column, add the RA3 of 350 μ l, place the centrifugal 2min of 2min, 1.2 ten thousand rpm, outwell lower floor's solution.F) in adsorption column, add the RA3 of 300 μ l, place the centrifugal 2min of 2min, 1.2 ten thousand rpm, outwell lower floor's solution.G) after opening the lid 3min of adsorption column, 1.2 ten thousand rpm are empty from 2min, outwell lower floor's solution, repeat this step once.H) sky is after end, and adsorption column is transferred in corresponding centrifuge tube, opens adsorption column lid and places 15min, ethanol is volatilized completely, adds 60 μ l Rnase-H in adsorption column
2o (carrying in test kit) wash-out, places the centrifugal 2min of 2min, 1.2 ten thousand rpm.I) centrifugal gained template is sucked back in adsorption column, place the centrifugal 2min of 2min, 1.2 ten thousand rpm, repeat this step twice.J) lose adsorption column, the template in centrifuge tube is taken out 2 μ l, measure its concentration with Nano drop, and record its concentration and 260/280,260/230 ratio.
Two, for detecting sensitivity and the specificity analyses of the test kit of Respirovirus
1, the reference material Template preparation of different concns
By each RNA template (the 16 kinds of recombinant plasmids built in corresponding step one 1) all calculation in quantities to 10 after above-mentioned steps one purifying
10copy/μ l, and gradient dilution, obtain 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the template dilution of the different concns such as copy/μ l, 10 copy/μ l.
2, reaction system preparation
Get 15 μ l constant-temperature amplification damping fluids (see embodiment 1 step 1), 10 μ l constant-temperature amplification enzyme solution (see embodiment 1 step 2), 25 μ l concentration template dilution be mixed into 50 μ l reaction solns, the 24 reaction chamber dish-style chips (see embodiment 1 step 3) being mounted with primer pair and ssDNA probe are injected, Quick spin 30s after sealing membrana oralis after vortex concussion evenly.
3, isothermal amplification reactions and detection
Dish-style chip is placed in rich Isochip-RT constant-temperature amplification instrument difficult to understand, 41 DEG C of reactions 1 hour is set, and complete real-time fluorescence scanning simultaneously.And according to the method for embodiment 1 step 23, result is judged.
Three, result
Result is as shown in Figure 2:
(1) for the RNA sample of recombinant plasmid pUC19-InfA, the 1# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 2#-16# reaction chamber is all without amplification curve, be shown as feminine gender.
(2) for the RNA sample of recombinant plasmid pUC19-InfA_H1, the 2# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 1#, 3#-16# reaction chamber is all without amplification curve, be shown as feminine gender.
(3) for the RNA sample of recombinant plasmid pUC19-InfA_H3, the 3# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 1#, 2# and 4-16# reaction chamber is all without amplification curve, be shown as feminine gender.
(4) for the RNA sample of recombinant plasmid pUC19-InfB, the 4# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 1#-3# and 5#-16# reaction chamber is all without amplification curve, be shown as feminine gender.
(5) for the RNA sample of recombinant plasmid pUC19-RSV, the 5# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 1#-4# and 6#-16# reaction chamber is all without amplification curve, be shown as feminine gender.
(6) for the RNA sample of recombinant plasmid pUC19-HRV, the 6# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 1#-5# and 7#-16# reaction chamber is all without amplification curve, be shown as feminine gender.
(7) for the RNA sample of recombinant plasmid pUC19-ADV, the 7# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 1#-6# and 8#-16# reaction chamber is all without amplification curve, be shown as feminine gender.
(8) for the RNA sample of recombinant plasmid pUC19-PIV1, the 8# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 1#-7# and 9#-16# reaction chamber is all without amplification curve, be shown as feminine gender.
(9) for the RNA sample of recombinant plasmid pUC19-PIV2, the 9# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 1#-8# and 10#-16# reaction chamber is all without amplification curve, be shown as feminine gender.
(10) for the RNA sample of recombinant plasmid pUC19-PIV3, the 10# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 1#-9# and 11#-16# reaction chamber is all without amplification curve, be shown as feminine gender.
(11) for the RNA sample of recombinant plasmid pUC19-Corn_OC, the 11# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 1#-10# and 12#-16# reaction chamber is all without amplification curve, be shown as feminine gender.
(12) for the RNA sample of recombinant plasmid pUC19-Corn_NL, the 12# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 1#-11# and 13#-16# reaction chamber is all without amplification curve, be shown as feminine gender.
(13) for the RNA sample of recombinant plasmid pUC19-hMPV, the 13# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 1#-12# and 14#-16# reaction chamber is all without amplification curve, be shown as feminine gender.
(14) for the RNA sample of recombinant plasmid pUC19-Entv, the 14# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 1#-13# and 15#-16# reaction chamber is all without amplification curve, be shown as feminine gender.
(15) for the RNA sample of recombinant plasmid pUC19-EV71, the 15# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 1#-14# and 16# reaction chamber is all without amplification curve, be shown as feminine gender.
(16) for the RNA sample of recombinant plasmid pUC19-CA16, the 16# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 1#-15# reaction chamber is all without amplification curve, be shown as feminine gender.
In addition, for the RNA sample of all 16 kinds of recombinant plasmids, the 17# reaction chamber (internal reference) of 24 reaction chamber dish-style chips and 18# reaction chamber (negative control) are all without amplification curve.Why 17# reaction chamber (internal reference) is because sample to be tested is the RNA nucleic acid of 16 kinds of recombinant plasmids without amplification curve, the RNA not containing house-keeping gene GAPDH in human genome.
These results suggest that this test kit has higher amplification sensitivity and specific amplification.
The detection of embodiment 3, actual clinical sample
One, clinical sample type
The present embodiment adopt clinical sample from Shenzhen San Yuan in the Nasopharyngeal swabs sample (in line with the principle that this picker is voluntary) gathered, swab of adopting is deposited in 3mL physiological saline, and totally 560 is routine.
Two, the extraction of viral nucleic acid in clinical sample
It is QIAamp Viral RNA Mini Kit (Qiagen) that clinical sample viral nucleic acid extracts test kit used, extracts as follows:
(1) get in step one deposit clinical sample swab physiological saline 140 μ l in 1.5ml centrifuge tube;
(2) add 560 μ l to contain the Buffer AVL of Carrier RNA mixed solution (namely 5.6 μ l Carrier RNA mixed solution+560 μ lBuffer AVL are in centrifuge tube, slight whirlpool 15s;
(3) after brief centrifugation completes, room temperature (15-25 DEG C) places 10-20min, to ensure that Buffer AVL has the sufficient time to carry out cracking in centrifuge tube;
(4) 560 μ l ethanol (96-100%, volumn concentration) are added in centrifuge tube, whirlpool mixing 15s, centrifugal;
(5) get 630 μ l sample solutions in adsorption column, place 2min, make it fully contact with adsorption column, 8000rpm, centrifugal 1min, change clean collection tube;
(6) if sample solution is more than 630 μ l, then previous step is repeated;
(7) in adsorption column, add the AW1 washing lotion of 500 μ l, place 2min, make it fully contact with adsorption column, the centrifugal 1min of 8000rpm, changes clean collection tube;
(8) in adsorption column, add the AW2 washing lotion of 500 μ l, place 2min, make it fully contact with adsorption column, the centrifugal 3min of 12000rpm, changes clean collection tube;
(9) in adsorption column, add the AW2 washing lotion of 300 μ l, place 2min, make it fully contact with adsorption column, the centrifugal 3min of 12000rpm, changes clean collection tube;
(10) adsorption column lid is opened placement 2min, make pressure inside and outside it consistent, 8000rpm is empty from 1min.Empty after end, repeat this step once.
(11) sky is after end, is transferred to by adsorption column in 1.5ml centrifuge tube, opens adsorption column lid, places 20-30min, makes ethanol volatilize completely;
(12) add 50 μ l Buffer AVE wash-out nucleic acid, the centrifugal 1min of 8000rpm, centrifugal complete after, the nucleic acid under wash-out is sucked back adsorption column, after centrifugal 1min, loses adsorption column.
Three, the detection of actual clinical sample
1, reaction system preparation
Get 15 μ l constant-temperature amplification damping fluids (see embodiment 1 step one 1), 10 μ l constant-temperature amplification enzyme solution (see embodiment 1 step one 2), 25 μ l step 2 extract the clinical sample solution obtained and be mixed into 50 μ l reaction solns, the 24 reaction chamber dish-style chips (see embodiment 1 step one 3) being mounted with primer pair and ssDNA probe are injected, Quick spin 30s after sealing membrana oralis after vortex concussion evenly.
2, isothermal amplification reactions and detection
Dish-style chip is placed in rich Isochip-RT constant-temperature amplification instrument difficult to understand, 41 DEG C of reactions 1 hour is set, and complete real-time fluorescence scanning simultaneously.And according to the method for embodiment 1 step 23, result is judged.
Experiment arranges the reliability demonstration of conventional RT-PCR method for test kit detected result of the present invention simultaneously.Specificity RT-PCR primer sequence for detecting each Respirovirus is as shown in table 1.
Table 1 is for detecting the specificity RT-PCR primer sequence of 16 kinds of Respiroviruses
| Index name | RT-PCR upstream primer (5 '-3 ') | RT-PCR downstream primer (5 '-3 ') |
| Influenza A virus | CTTGTCTTTAGCCAYTCCATGAGAGC | CTAACCGAGGTCGAAACGTAYGTTCT |
| Influenza A virus H1 hypotype | CATCTAYCATTCCAGTCCACCCCCCT | ACCCCAGAAATAGCHAAAAGACCCAA |
| Influenza A virus H3 hypotype | GTGCTITTAATCAAAAGTATGTCTCCCG | CACAGTITCTACCAAAAGAAGCCAAC |
| Influenza B virus | TTCAGGTACATGACCATGAGACARTA | TYGGTGGGAAAGAATTTGACCTAGAC |
| Parainfluenza virus I type | GTAGTCTCATTCACAGTGGGYAAGGA | ATCTCATTATTACCYGGACCAAGTCT |
| Parainfluenza virus II type | TACAAGACACAACCTCCTGGTATAGCA | GGACGCCTAAATATGGACCTCTCCT |
| Parainfluenza virus type III | TCTCTCTGTGTTTTCCCVGGACACCC | GACTTAAATCCYAGGATCTCTCATAC |
| HCoV-HKU1/OC43 | AGGCTGTATGATGAATGTTGCTAAG | CCTGCACCTAAATGYAAAACWCGCATAT |
| HCoV-229E/NL63 | CAATACAGGGTCCTCCTGGTAGTGG | CACTACCAGGAGGACCCTGTATTGT |
| Respiratory syncytial virus | ACTGATCCTGCATTITCACARTACCA | AGGTGTAACIACACCTITAAGCACTTA |
| Rhinovirus | TCYAGCCTGCGTGGCTGCCTGC | GAAACACGGACACCCAAAGTAGTYGGT |
| Adenovirus | TGYAACATGACCAARGACTGGTTC | GAAGGGTGGRCTCRTCCATGGGCTC |
| Human metapneumovirus | GCAACATTGAAYTGATCYTCAGGAA | ACCCRTGCAAAGTYAGCACAGGAAG |
| Enterovirus | CCCCTGAATGCGGCTAATCCYAAC | CCAATCCAATAGCTATATGGYAACAA |
| Enterovirus EV 71 type | AACACCCGTAYGTGCTTGATGCTGG | TAGGGATTACTGGCGTCGCTCCTTG |
| Enterovirus CA16 type | CACCCCCATGCARCGCTTGTGYTTT | TRGACTGCCACGGRCCATCTCTTCC |
Four, result
The detected result display of test kit of the present invention, for each Respirovirus, adopts the sample example of the RT-PCR method detection positive to adopt test kit of the present invention to detect and all shows positive findings.In addition, for a few sample example, adopt RT-PCR method to detect negative, but adopt test kit detected result of the present invention to be positive.Detecting by carrying out later stage resampling RT-PCR to these sample examples, finding that these sample examples are detected as corresponding Respirovirus really positive.More than show, test kit of the present invention is to the recall rate of corresponding Respirovirus higher than RT-PCR method, and its detected result accurately and reliably.Test kit of the present invention and RT-PCR method detect the statistics of common respiratory tract virus clinical sample see table 2.
Table 2 test kit of the present invention and RT-PCR method detect the statistics of common respiratory tract virus clinical sample
Claims (10)
1., for detecting the primer pair group of Respirovirus, be made up of following 16 primer pairs:
The primer pair 1 be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2;
The primer pair 2 be made up of two single strand dnas shown in sequence in sequence table 3 and sequence 4;
The primer pair 3 be made up of two single strand dnas shown in sequence in sequence table 5 and sequence 6;
The primer pair 4 be made up of two single strand dnas shown in sequence in sequence table 7 and sequence 8;
The primer pair 5 be made up of two single strand dnas shown in sequence in sequence table 9 and sequence 10;
The primer pair 6 be made up of two single strand dnas shown in sequence in sequence table 11 and sequence 12;
The primer pair 7 be made up of two single strand dnas shown in sequence in sequence table 13 and sequence 14;
The primer pair 8 be made up of two single strand dnas shown in sequence in sequence table 15 and sequence 16;
The primer pair 9 be made up of two single strand dnas shown in sequence in sequence table 17 and sequence 18;
The primer pair 10 be made up of two single strand dnas shown in sequence in sequence table 19 and sequence 20;
The primer pair 11 be made up of two single strand dnas shown in sequence in sequence table 21 and sequence 22;
The primer pair 12 be made up of two single strand dnas shown in sequence in sequence table 23 and sequence 24;
The primer pair 13 be made up of two single strand dnas shown in sequence in sequence table 25 and sequence 26;
The primer pair 14 be made up of two single strand dnas shown in sequence in sequence table 27 and sequence 28;
The primer pair 15 be made up of two single strand dnas shown in sequence in sequence table 29 and sequence 30;
The primer pair 16 be made up of two single strand dnas shown in sequence in sequence table 31 and sequence 32.
2., for detecting the complete single stranded DNA of Respirovirus, be made up of probe groups and primer pair group according to claim 1;
Described probe groups is made up of following 16 ssDNA probe: the ssDNA probe 1 in sequence table shown in sequence 33; SsDNA probe 2 in sequence table shown in sequence 34; SsDNA probe 3 in sequence table shown in sequence 35; SsDNA probe 4 in sequence table shown in sequence 36; SsDNA probe 5 in sequence table shown in sequence 37; SsDNA probe 6 in sequence table shown in sequence 38; SsDNA probe 7 in sequence table shown in sequence 39; SsDNA probe 8 in sequence table shown in sequence 40; SsDNA probe 9 in sequence table shown in sequence 41; SsDNA probe 10 in sequence table shown in sequence 42; SsDNA probe 11 in sequence table shown in sequence 43; SsDNA probe 12 in sequence table shown in sequence 44; SsDNA probe 13 in sequence table shown in sequence 45; SsDNA probe 14 in sequence table shown in sequence 46; SsDNA probe 15 in sequence table shown in sequence 47; SsDNA probe 16 in sequence table shown in sequence 48.
3. complete single stranded DNA according to claim 2, is characterized in that: 5 ' end of the every bar ssDNA probe in described probe groups is all marked with fluorescent reporter group FAM, and 3 ' end is all marked with fluorescent quenching group TAMRA.
4. for detecting the test kit of Respirovirus, containing the complete single stranded DNA described in primer pair group according to claim 1 or Claims 2 or 3.
5. test kit according to claim 4, is characterized in that: described test kit is also containing internal reference primer pair and internal reference probe; Described internal reference primer pair is made up of two single strand dnas shown in sequence in sequence table 49 and sequence 50; Described internal reference probe is the ssDNA probe shown in sequence in sequence table 51;
Concrete, 5 ' end of described internal reference probe is marked with fluorescent reporter group FAM, and 3 ' end is marked with fluorescent quenching group TAMRA.
6. the test kit according to claim 4 or 5, is characterized in that: also containing constant-temperature amplification damping fluid and constant-temperature amplification enzyme solution in described test kit;
The solvent of described constant-temperature amplification damping fluid is water, solute and concentration as follows: the Tris-HCL of 200mM pH 8.0,50mM DTT, 10mM dNTP, 10mM rNTP, 80mM MgCl
2, 450mM KCl, 15% volumn concentration DMSO, 1M sorbyl alcohol, 20mM tetramethyl ammonium chloride;
The solvent of described constant-temperature amplification enzyme solution is water, solute and concentration as follows: AMV reversed transcriptive enzyme 1U/ μ l, T7RNA polysaccharase 5U/ μ l, ribonuclease H 0.5U/ μ l, Pyrophosphate phosphohydrolase 0.5U/ μ l, RNA enzyme inhibitors 5U/ μ l, BSA 0.5 μ g/ μ l.
7. according to described test kit arbitrary in claim 4-6, it is characterized in that: also containing dish-style chip in described test kit;
1# to the 18# reaction chamber of described dish-style chip deposits dry following (1)-(18) respectively:
(1) primer pair 1 described in claim 1 and the ssDNA probe described in claim 21;
(2) primer pair 2 described in claim 1 and the ssDNA probe described in claim 22;
(3) primer pair 3 described in claim 1 and the ssDNA probe described in claim 23;
(4) primer pair 4 described in claim 1 and the ssDNA probe described in claim 24;
(5) primer pair 5 described in claim 1 and the ssDNA probe described in claim 25;
(6) primer pair 6 described in claim 1 and the ssDNA probe described in claim 26;
(7) primer pair 7 described in claim 1 and the ssDNA probe described in claim 27;
(8) primer pair 8 described in claim 1 and the ssDNA probe described in claim 28;
(9) primer pair 9 described in claim 1 and the ssDNA probe described in claim 29;
(10) primer pair 10 described in claim 1 and the ssDNA probe described in claim 2 10;
(11) primer pair 11 described in claim 1 and the ssDNA probe described in claim 2 11;
(12) primer pair 12 described in claim 1 and the ssDNA probe described in claim 2 12;
(13) primer pair 13 described in claim 1 and the ssDNA probe described in claim 2 13;
(14) primer pair 14 described in claim 1 and the ssDNA probe described in claim 2 14;
(15) primer pair 15 described in claim 1 and the ssDNA probe described in claim 2 15;
(16) primer pair 16 described in claim 1 and the ssDNA probe described in claim 2 16;
(17) the internal reference primer pair described in claim 5 and described internal reference probe;
(18) negative controls.
8. in claim 1-7 arbitrary described primer pair group or complete single stranded DNA or test kit in the non-diagnostic object application detected or in auxiliary detection Respirovirus.
9. application according to claim 8, it is characterized in that: in described application, by constant-temperature amplification enzyme solution described in constant-temperature amplification damping fluid described in 15 μ l and 10 μ l, be injected in described dish-style chip after mixing with 25 μ l sample to be tested solution, isothermal reaction 1h at 41 DEG C.
10., according to arbitrary described primer pair group in 1-9 or complete single stranded DNA or test kit or application, it is characterized in that: described Respirovirus be following at least one: influenza A virus, influenza A virus H1 hypotype, influenza A virus H3 hypotype, Influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, parainfluenza virus I type, parainfluenza virus II type, parainfluenza virus type III, HCoV-HKU1/OC43, HCoV-229E/NL63, human metapneumovirus, enterovirus, enterovirus EV 71 type and enterovirus CA16 type.
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