CN108165667A - The triple real-time fluorescence quantitative RT-PCR detection reagent kits of human respiratory syncytial virus - Google Patents
The triple real-time fluorescence quantitative RT-PCR detection reagent kits of human respiratory syncytial virus Download PDFInfo
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- CN108165667A CN108165667A CN201711266806.5A CN201711266806A CN108165667A CN 108165667 A CN108165667 A CN 108165667A CN 201711266806 A CN201711266806 A CN 201711266806A CN 108165667 A CN108165667 A CN 108165667A
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Classifications
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- C—CHEMISTRY; METALLURGY
- 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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- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/68—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
- C12Q1/6851—Quantitative amplification
Abstract
The present invention provides a kind of triple real time fluorescent quantitative RT PCR detection kits of human respiratory syncytial virus, it is related to universal, A types and Type B, it is made of quantitative PCR reaction solution, HRSV standard items, HRSV negative controls, HRSV positive reference substances, specification and box body, wherein quantitative PCR reaction solution contains PCR buffer solutions, MgCl2, dNTPs, RNA reverse transcriptase, hot resistant DNA polymerase, three pairs of upstream and downstream primers, the universal probes of HRSV, HRSVA types probe and HRSVB type probes.Using kit of the present invention, parting detection can not only be carried out to the HRSV in sample, and real-time accurate quantitative analysis can be carried out to positive-virus, sensitivity is up to 1.0 × 101Copy/μ L saves production and testing cost, improves detection efficiency, meets clinical diagnosis demand, help to formulate targeted treatment schemes in time.
Description
Technical field
The invention belongs to biotechnologies, are related to fluorescence quantitative RT-PCR detecting kit, and in particular to a kind of three probes
Human respiratory syncytial virus in real-time fluorescence quantitative RT-PCR detection patient bronchoalveolar lavage fluid, saliva, throat swab equal samples
(HRSV) novel agent box, and can Classification Identification and quantitative detection be carried out to HRSV simultaneously.
Background technology
Respiratory Syncytial Virus(RSV) (RSV) is moved in the experiment by Morris and Yates from an only cold symptoms in 1956
It is isolated in the nasopharyngeal secretions of object chimpanzee.Then in nineteen fifty-seven Chanock etc. successively from 2 points of Baltimore cities
It does not suffer from an inflammation of the lungs and has in the throat swab of wheezing symptoms infant and be separated to, because it can form special cell fusion in tissue cultures
Lesion and gain the name.RSV belongs to paramyxovirus section pneumonitis virus category, is the tunicary RNA virus of sub-thread minus strand, nucleic acid overall length
About 15kb can be divided into two kinds of hypotypes of A, B.
RSV is a kind of one of Respirovirus generally popular in current human population worldwide, infection morbidity in global,
Usually in winter, spring outbreak of epidemic, popular region is wide, and explosion time is long, it has also become worldwide public health problem.RSV is passed
The mode broadcast is mainly by droplet transmission, secondly the finger of pollution directly draws virus inoculation to schneiderian membrane and eye mucosa
Play infection.Clinical symptoms are low-heat, sneeze, runny nose, cough etc., and rsv infection is common in less than 5 years old children, old man and immunity
Decline crowd such as organ transplant person etc. is infant's capillary bronchitis and the most common cause of disease of pneumonia, seriously endangers infant
Health and the reason of cause one of Children and teenager asthma extremely important.Lasting immunity cannot be obtained after rsv infection
Power, therefore, superinfection are very common, and if Nosocomial rsv infection, the caused infant death rate compares Community-acquired
RSV infects higher.One shows the meta-analysis of global less than 5 years old Acute Lower Respiratory Infection in Children：Estimation every year by
It is infected caused by RSV up to 3.38 × 107Example, wherein at least 3.4 × 1O6Example infant needs hospitalization, and estimation can cause 66000--
199000 infant death, and 99% developing country is happened in dead infant, which results in the warps that developing country is heavy
The high mortality of Ji burden and high risk child.Therefore, quick, sensitive, special RSV test in laboratory methods are established, to disease
Early diagnosis, treatment and prevention and control have great importance.
However RSV clinical detections are more difficult, virus purification cell culture method is considered as separation and identification virus always
" goldstandard ", but since cell culture condition is stringent, need the time long, positive rate is low, and sample size requirements to be checked compared with
Height can not detect trace sample, parting difficulty, therefore this method is not suitable for clinical RSV quickly detection and parting.Immunology
Detection is also a kind of method of quick diagnosis infectious diseases, but since its sensitivity is less than cell culture, therefore clinical labororatory
Also seldom selection this method detects for the diagnosis of RSV.In addition, the new virus detection method found in recent years is also efficient high
Accuracy RSV context of detection has carried out a variety of trials, such as Surface enhanced Raman scattering detection, atomic force microscope technology.But
Due to its for instrument and equipment, technical staff it is more demanding, make it extensively using still acquiring a certain degree of difficulty.Therefore, it is badly in need of exploitation
A kind of quick, easy, sensitive and special method is used for the detection of clinical rsv infection case.
With the rapid development of Protocols in Molecular Biology, polymerase chain reaction (polymerasechainreaction,
PCR) technology, the real-time fluorescence quantitative RT-PCR particularly risen in recent years provide new side for the detection of pathogenic microorganism
To.The basic principle of real-time fluorescence quantitative RT-PCR is 5 ' -3 ' the excision enzyme nucleic acids activities using Taq enzyme, in regular-PCR
On the basis of design double immunofluorescense probe, both ends difference mark fluorescent reporter group (R) and quenching group (Q)；Probe has been kept
The fluorescence signal of R groups is inhibited by Q groups when whole, once probe is cut off, the inhibiting effect of Q groups disappears, R group it is glimmering
Optical signal can be detected.The technology is by realizing the detection of fluorescence signal in the real-time prison to product amount during RT-PCR
It surveys, and can accurately calculate original template amount, in addition to having many advantages, such as that quantitatively accurate, detection is quick, maximum advantage is to have used
Fully closed pipe detection, eliminates the post processing to PCR product, avoids cross contamination.
Current Real-Time Fluorescent Quantitative PCR Technique is with the further development of material and instrument, by fluorescence probe
5 ' ends mark different fluorescent dyes (FAM, JOE, TAMRA etc.) and multichannel fluorescence quantitative PCR instrument, can realize gene point
The researchs such as type, detection in Gene Mutation, snp analysis.
Invention content
The object of the present invention is to provide a kind of triple real-time fluorescence quantitative RT-PCR detection reagents of human respiratory syncytial virus
Box is a kind of while detects HRSV and carries out triple real-time fluorescence quantitative RT-PCR kits of A types and Type B parting, by quantitative
PCR reaction solutions, HRSV standard items, HRSV negative controls, HRSV positive reference substances, specification and box body composition, wherein quantitative
PCR reaction solution contains PCR buffer solutions, MgCl2, dNTPs, RNA reverse transcriptase, hot resistant DNA polymerase, 3 pairs of upstream and downstream primers,
The universal probes of HRSV, HRSV A types probes and HRSV Type B probes.
Universal HRSV detection primers and probe：
HRSV1：5'-GCAAATATGGAAACATACGTGAA-3'
HRSV2：5'-ACCCATATTGTWAGTGATGCAG-3'
Probe-HRSV7：5'-FAM-CTTCACGAAGGCTCCACATACACAGC-BHQ1-3'；
HRSV A types detection primers and probe：
HRSV3：5'-CAACTTCTGTCATCCAGCAA-3'
HRSV4：5'-GCACATCATAATTAGGAGTATCAAT-3'
Probe-HRSV8：5'-JOE-ACACCATCCAACGGAGCACAGGAGATA-BHQ1-3'；
HRSV Type Bs detection primer and probe：
HRSV5：5'-GATGCAAATCATAAATTCACAGGA-3'
HRSV6：5'-TCCAGCATCTTTAAGTATCTTTATAGT-3'
Probe-HRSV9：5'-TAMRA-GGTATGTTATATGCTATGTCCAGGTTAGGAAGGG-BHQ2-3'；
Wherein, W is A or T.
HRSV standard items sequences are：
atccgccaatcagccaaacagccaacaaaacaaccagccaatccaaaaccagccacctggaaaaaatcgacaatata
gttacaaaaaaagaaaagggtggggcaaatatggaaacatacgtgaacaagcttcacgaaggctccacatacacagc
tgctgttcaatacaatgtcctagaaaaagacgatgaccctgcatcacttacaatatgggtgcccatgttccaatcat
ctatgccagcagatttacttataaaagaactagctaatgtcaacatactagtgaaacaaatatccacacccaaggga
ccttca。
HRSV negative controls are sterile water for injection sample, and HRSV positive reference substances are HRSV inactivation of viruses strain samples.
Kit of the present invention should be stored in -20 DEG C, reduce multigelation to the greatest extent.
Kit application method of the present invention：
Detection should all set up positive control and negative control every time, and standard items are diluted to 1 × 10 with aseptic deionized water2
1×106Copy.
The extraction of human respiratory syncytial virus RNA：It operates in strict accordance with the Trizol extraction agents of commercialization, is marked from clinic
Human respiratory syncytial virus RNA is extracted in this or virus purification culture.
PCR amplification detects：Three colors (or more) carry out on fluorescence quantitative PCR instrument, the total volume of each PCR reaction systems
For 20ul, including 18ulPCR reaction solutions and 2ul RNA templates.Probe in detecting pattern is：FAM, JOE and TAMRA channel point
HRSV universal, HRSVA types and HRSVB types Yong Yu not detected.
Pcr amplification reaction condition：42 DEG C of 5 minutes reverse transcription RNA, 95 DEG C of 2 minutes pre-degenerations, 95 DEG C 10 seconds → 55 DEG C 40
Second, totally 40 recycle.After being provided with, save file runs program.
PCR reaction systems are calculated as with 20 μ l：Primer HRSV1, HRSV2, HRSV3, HRSV4, HRSV5, HRSV6 final concentration is each
For 0.4 μm of ol/L, Probe-HRSV7 final concentration of 0.2 μm of ol/L, Probe-HRSV8 final concentration of 0.4 μm of ol/L, Probe-
HRSV9 final concentration of 0.4 μm of ol/L, 2.5mmol/L dNTPs 2 μ L, MgCl20.8 μ L, 10 × ExTaq enzyme buffer liquid, 2 μ L,
0.4 0.4 μ L, RNA template of μ L, 5U/ μ LExTaqDNA polymerases of RNA reverse transcriptase, 2 μ L, deionized water complement to 20 μ of total volume
L。
Fluorescent quantitation result is reported：1. the amplification curve of sample is detected without increased logarithmic phase or three probe CT
(thresholdcycle) value is >=40 feminine gender；2. it detects sample F AM probe CT value≤38 and amplification curve has apparent logarithm
Rise period then illustrates human respiratory syncytial virus for the positive；JOE probes CT value≤38 and amplification curve has in same reaction system
Apparent increased logarithmic phase, then it is A type strains to illustrate human respiratory syncytial virus, and TAMRA probe CT value≤38 and amplification curve have bright
Aobvious increased logarithmic phase then illustrates human respiratory syncytial virus for Type B strain, if JOE and TAMRA probes CT values in same reaction system
≤ 38 and amplification curve has apparent increased logarithmic phase, then illustrates human respiratory syncytial virus for A, Type B strain mixed infection；3. it examines
The a certain 38 ＜ CT values ＜ 40 of probe of sample need to re-start sample RNA extractions and RT-PCR detections.
The present invention has been developed using real-time fluorescence quantitative RT-PCR and three color specificity fluorescent probes for people
Respiratory Syncytial Virus(RSV) quantifies and the kit of parting detection.The invention kit is to the human respiratory syncytial disease in clinical sample
Poison can not only carry out parting detection, and can carry out real-time accurate quantitative analysis to positive-virus, and existing single currently on the market
Virus detection kit is compared, and has not only saved production cost and testing cost, also improves detection efficiency, can meet people's breathing
The clinical diagnosis demand of road syncytial virus helps to formulate targeted treatment schemes in time.Using kit of the present invention, to sample
In HRSV can not only carry out parting detection, and real-time accurate quantitative analysis can be carried out to positive-virus, sensitivity is up to 1.0 × 101
Copy/μ L saves production and testing cost, improves detection efficiency.The present invention is based on current technical backgrounds, design and are exhaled for people
The triple fluorescent quantitative RT-PCR detection for inhaling the specific probe of road syncytial virus, exploitation energy while parting and the quantitative virus tries
Agent box, it is intended to meet the needs of clinical human respiratory syncytial virus infects early stage, quick diagnosis, for clinical human respiratory syncytial disease
The epidemiological survey of poison infection and immunotherapy targeted autoantibody provide help.
Description of the drawings
Fig. 1 is the amplification curve of HRSV concentration gradient standard items.1~7 corresponding plasmid concentration is 1.0 × 107,
1.0× 106,1.0×105,1.0×104,1.0×103,1.0×102,1.0×101Copy/μ L；8 be negative control.
Fig. 2 is the standard curve of HRSV concentration gradient standard items.
Fig. 3 is human respiratory syncytial virus's universal real time fluorescent quantitative RT-PCR detecting method amplification curve；Wherein, 1
For the universal amplification curves of HRSV；2~10 be chlamydia trachomatis (CT), mycoplasma pneumoniae (MP), Ureaplasma urealyticum (UU), people
Cytomegalovirus (HCMV), Epstein-Barr virus (EB), herpes simplex virus type II (HSV II), measles virus (MV), mumps virus
(MuV) and human genome DNA's amplification curve, 11 be negative control amplification curve.
Fig. 4 is human respiratory syncytial virus's A type real-time fluorescence quantitative RT-PCR detection method amplification curves；Wherein, 1 is
HRSV A type amplification curves；；2~10 be chlamydia trachomatis (CT), mycoplasma pneumoniae (MP), Ureaplasma urealyticum (UU), people are big and small
Cellular virus (HCMV), Epstein-Barr virus (EB), herpes simplex virus type II (HSV II), measles virus (MV), mumps virus (MuV)
It is negative control amplification curve with human genome DNA's amplification curve, 11.
Fig. 5 is human respiratory syncytial virus's Type B real-time fluorescence quantitative RT-PCR detection method amplification curve；Wherein, 1 is
HRSV Type B amplification curves；2~10 be chlamydia trachomatis (CT), mycoplasma pneumoniae (MP), Ureaplasma urealyticum (UU), human cytomegalovirus
Viral (HCMV), Epstein-Barr virus (EB), herpes simplex virus type II (HSV II), measles virus (MV), mumps virus (MuV) and
Human genome DNA's amplification curve, 11 be negative control amplification curve.
Specific embodiment
The present invention is described further in conjunction with the embodiments with attached drawing.It should be understood that these embodiments are for illustration purposes only,
Rather than it limits the scope of the invention.
Embodiment 1
A kind of human respiratory syncytial virus provided by the invention is universal, A types and the triple real-time fluorescence quantitative RT-PCRs of Type B
Detection kit is made of quantitative PCR reaction solution, HRSV standard items, HRSV negative controls, HRSV positive reference substances.Wherein
Quantitative PCR reaction solution contains PCR buffer solutions, MgCl2, dNTPs, RNA reverse transcriptase, hot resistant DNA polymerase, 3 pairs of upstream and downstream draw
The universal probe of object, HRSV, HRSV A types probes and HRSV Type B probes.Negative controls are sterile water for injection sample,
HRSV positive reference substances are HRSV inactivation of viruses strain samples.
Universal HRSV detection primers and probe：
HRSV1：5'-GCAAATATGGAAACATACGTGAA-3'
HRSV2：5'-ACCCATATTGTWAGTGATGCAG-3'
Probe-HRSV7：5'-FAM-CTTCACGAAGGCTCCACATACACAGC-BHQ1-3'；
HRSV A types detection primers and probe：
HRSV3：5'-CAACTTCTGTCATCCAGCAA-3'
HRSV4：5'-GCACATCATAATTAGGAGTATCAAT-3'
Probe-HRSV8：5'-JOE-ACACCATCCAACGGAGCACAGGAGATA-BHQ1-3'；
HRSV Type Bs detection primer and probe：
HRSV5：5'-GATGCAAATCATAAATTCACAGGA-3'
HRSV6：5'-TCCAGCATCTTTAAGTATCTTTATAGT-3'
Probe-HRSV9：5'-TAMRA-GGTATGTTATATGCTATGTCCAGGTTAGGAAGGG-BHQ2-3'；
Wherein, W is A or T.
HRSV standard items sequences are：
atccgccaatcagccaaacagccaacaaaacaaccagccaatccaaaaccagccacctggaaaaaatcgacaatata
gttacaaaaaaagaaaagggtggggcaaatatggaaacatacgtgaacaagcttcacgaaggctccacatacacagc
tgctgttcaatacaatgtcctagaaaaagacgatgaccctgcatcacttacaatatgggtgcccatgttccaatcat
ctatgccagcagatttacttataaaagaactagctaatgtcaacatactagtgaaacaaatatccacacccaaggga
ccttca。
Kit of the present invention should be stored in -20 DEG C, reduce multigelation to the greatest extent.
Pcr amplification reaction condition：42 DEG C of 5 minutes reverse transcription RNA, 95 DEG C of 2 minutes pre-degenerations, 95 DEG C 10 seconds → 55 DEG C 40
Second, totally 40 recycle.After being provided with, save file runs program.
PCR reaction systems are calculated as with 20 μ l：Primer HRSV1, HRSV2, HRSV3, HRSV4, HRSV5, HRSV6 final concentration is each
For 0.4 μm of ol/L, Probe-HRSV7 final concentration of 0.2 μm of ol/L, Probe-HRSV8 final concentration of 0.4 μm of ol/L, Probe-
HRSV9 final concentration of 0.4 μm of ol/L, 2.5mmol/L dNTPs 2 μ L, MgCl20.8 μ L, 10 × ExTaq enzyme buffer liquid, 2 μ L,
0.4 0.4 μ L, RNA template of μ L, 5U/ μ LExTaqDNA polymerases of RNA reverse transcriptase, 2 μ L, deionized water complement to 20 μ of total volume
L。
2 human respiratory syncytial virus of embodiment is universal, the sensibility of A types and the triple detection kits of Type B, specificity,
Stability and repetitive test
(1) material：
Pathogenic microorganism is chosen to include：HRSV positive controls：Long plants of HRSV by attached children's hospital Zhao of Zhejiang University just
Say that scientific research group provides；Control group：It is chlamydia trachomatis, mycoplasma pneumoniae, Ureaplasma urealyticum, human cytomegalovirus, Epstein-Barr virus, simple
HerpesvirusⅡtype, measles virus, mumps virus and human genome respectively by the attached children's hospital's Bacteriology Room of Zhejiang University,
Viral Laboratory and gene magnification room provide.
(2) instrument and reagent
Fluorescence quantitative PCR instrument：American AB I Products, model ABI7500；PCR amplification instrument:American AB I Products,
Model ABI9700；Labworks image acquisition and analysis software:U.S.'s Bio-Rad Products, model GelDocXR；Desk type high speed freeze from
Scheming:German Eppendorf Products, model 5804R；ExTaq archaeal dna polymerases, dNTPs, RNA reverse transcriptase, PCR delay
Fliud flushing, MgCl2, enzyme inhibitor etc. be purchased from precious bioengineering (Dalian) Co., Ltd, pUC57 carriers etc. purchased from Hangzhou Qing Ke Chinese catalpas
Prosperous Bioisystech Co., Ltd.
(3) design and synthesis of primer and probe：
Bioinformatic analysis is carried out to the sequence of HRSV, designs and screen the RT-PCR of HRSV universal, A types and Type B
Amplimer and specificity fluorescent probe, the synthesis of commission Hangzhou Qing Ke Zi Xi Bioisystech Co., Ltd.
Universal HRSV detection primers and probe：
HRSV1：5'-GCAAATATGGAAACATACGTGAA-3'
HRSV2：5'-ACCCATATTGTWAGTGATGCAG-3'
Probe-HRSV7：5'-FAM-CTTCACGAAGGCTCCACATACACAGC-BHQ1-3'；
A type HRSV detection primers and probe：
HRSV3：5'-CAACTTCTGTCATCCAGCAA-3'
HRSV4：5'-GCACATCATAATTAGGAGTATCAAT-3'
Probe-HRSV8：5'-JOE-ACACCATCCAACGGAGCACAGGAGATA-BHQ1-3'；
Type B HRSV detection primers and probe：
HRSV5：5'-GATGCAAATCATAAATTCACAGGA-3'
HRSV6：5'-TCCAGCATCTTTAAGTATCTTTATAGT-3'
Probe-HRSV9：5'-TAMRA-GGTATGTTATATGCTATGTCCAGGTTAGGAAGGG-BHQ2-3'；
Wherein, W is A or T.
(4) preparation of examination criteria product：
With the universal upstream and downstream amplimer amplification HRSV Strain of HRSV, amplified fragments are inserted into pUC57 clones after purification
Vector construction recombinant plasmid quantifies each plasmid with spectrophotometer, and each with 10 doubling dilutions with aseptic deionized water
Plasmid carries out fluorescence quantitative RT-RCR sensitivity tests.
(5) preparation of total serum IgE
The preparation of template ribonucleic acid：Using human respiratory syncytial virus's type strain cell culture fluid through inactivation as positive control, with
Normal cell culture solution extracts total serum IgE together with sample to be tested for negative control using Trizol methods.
Concrete operations are as follows：Positive control, negative control and each 200 μ L of sample to be tested are taken respectively in 1.5ml centrifuge tubes,
The Trizol for adding 500 μ L shakes 2-3min in vortice, adds in 140 μ L chloroforms, after centrifugation, supernatant is taken to be transferred to another
In 1.5ml centrifuge tubes, add 500 μ L isopropanol precipitatings, 75% ethyl alcohol washing precipitation is dry, finally with 20 μ LDEPC (pyrophosphoric acids two
Ethyl ester) water dissolution precipitation, in -20 DEG C of preservations.
(6) specific test of kit and sensitivity tests：
Using human respiratory syncytial virus is universal, A types and the triple detection kits of Type B to human respiratory syncytial virus into
Row detection, experimental group viral diagnosis result meet for positive and parting, control group chlamydia trachomatis, mycoplasma pneumoniae, solution urea branch
The inspections such as substance, human cytomegalovirus, Epstein-Barr virus, herpes simplex virus type II, measles virus, mumps virus and human genome
It is feminine gender to survey result, and specificity is 100% (Fig. 3-5).Detection sensitivity after the recombinant plasmid standard items doubling dilution of structure
Up to 10 copies；With 101、102、103、104、105、106、107Seven quantitative concentration gradient standard items make standard curve,
There is good linear relationship (Fig. 1-2) between CT values and plasmid copy number.
(7) stability and repetitive test of kit：
HRSV positive recombinant plasmids equal amount of mixture (the plasmid final concentration 1.0 that 10 times of 5 concentration are serially diluted respectively
× 106~1.0 × 102) triple real-time fluorescence quantitative RT-PCR amplifications are carried out, 4 repetitions of each series setting are divided into 2 batches
The secondary stability and repeatability for carrying out verification this method.The result shows that batch interior experiment coefficient of variation (CV) that repeats is up to
2.02%, experiment CV is repeated between batch and is up to 2.06%, the two CV values are 3% hereinafter, showing the human respiratory syncytial established
Viral universal, A types and three re-detection method of Type B have good stability and repeatability.Result of the test is shown in Table 1.
1 stability of table and repetitive test result
3 human respiratory syncytial virus of embodiment is universal, A types and the triple detection kits of Type B are in clinical sample detection
Application
(1) clinical sample detects：
Using human respiratory syncytial virus is universal, A types and the triple detection kits of Type B to 552 clinics suspected of breathing
The throat swab sample of road virus infection infant is detected, and simultaneous selection Shanghai Xingyao Medical Technology Development Co., Ltd. provides
, Yi Huo State Food and Drug Administrations approval Respirovirus detection kit (immunofluorescence technique) as control,
For evaluating the performance indicator of kit of the present invention.
(2) testing result：
Kit of the present invention detects 101 HRSV positive samples, wherein 53, HRSVA types altogether, and 48, HRSVB types are positive
Sample and the coincidence rate of contrast agents box testing result are 100%.Part test the results are shown in Table 2.
2 the method for the present invention of table and immunofluorescence technique part test results contrast
Present invention combination most preferred embodiment is described, however after the above for having read the present invention, ability
Field technique personnel can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims
The range of institute's limit.
Sequence table
<110>Zhejiang University
<120>The triple real-time fluorescence quantitative RT-PCR detection reagent kits of human respiratory syncytial virus
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>Artificial sequence (Unknown)
<400> 1
gcaaatatgg aaacatacgt gaa 23
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence (Unknown)
<400> 2
acccatattg twagtgatgc ag 22
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (Unknown)
<400> 3
caacttctgt catccagcaa 20
<210> 4
<211> 25
<212> DNA
<213>Artificial sequence (Unknown)
<400> 4
gcacatcata attaggagta tcaat 25
<210> 5
<211> 27
<212> DNA
<213>Artificial sequence (Unknown)
<400> 5
acaccatcca acggagcaca ggagata 27
<210> 6
<211> 24
<212> DNA
<213>Artificial sequence (Unknown)
<400> 6
gatgcaaatc ataaattcac agga 24
<210> 7
<211> 27
<212> DNA
<213>Artificial sequence (Unknown)
<400> 7
tccagcatct ttaagtatct ttatagt 27
<210> 8
<211> 34
<212> DNA
<213>Artificial sequence (Unknown)
<400> 8
ggtatgttat atgctatgtc caggttagga aggg 34
<210> 9
<211> 314
<212> DNA
<213>Artificial sequence (Unknown)
<400> 9
atccgccaat cagccaaaca gccaacaaaa caaccagcca atccaaaacc agccacctgg 60
aaaaaatcga caatatagtt acaaaaaaag aaaagggtgg ggcaaatatg gaaacatacg 120
tgaacaagct tcacgaaggc tccacataca cagctgctgt tcaatacaat gtcctagaaa 180
aagacgatga ccctgcatca cttacaatat gggtgcccat gttccaatca tctatgccag 240
cagatttact tataaaagaa ctagctaatg tcaacatact agtgaaacaa atatccacac 300
ccaagggacc ttca 314
Claims (5)
1. a kind of triple real-time fluorescence quantitative RT-PCR detection reagent kits of human respiratory syncytial virus, by quantitative PCR reaction solution,
HRSV standard items, HRSV negative controls, HRSV positive reference substances composition；It is characterized in that, quantitative PCR reaction solution contains PCR
Buffer solution, MgCl2, dNTPs, RNA reverse transcriptase, hot resistant DNA polymerase, three pairs of upstream and downstream primers, the universal probes of HRSV,
HRSVA types probe and HRSVB type probes；
Universal HRSV detection primers and probe：
HRSV1：5'-GCAAATATGGAAACATACGTGAA-3'
HRSV2：5'-ACCCATATTGTWAGTGATGCAG-3'
Probe-HRSV7：5'-FAM-CTTCACGAAGGCTCCACATACACAGC-BHQ1-3'；
HRSV A types detection primers and probe：
HRSV3：5'-CAACTTCTGTCATCCAGCAA-3'
HRSV4：5'-GCACATCATAATTAGGAGTATCAAT-3'
Probe-HRSV8：5'-JOE-ACACCATCCAACGGAGCACAGGAGATA-BHQ1-3'；
HRSV Type Bs detection primer and probe：
HRSV5：5'-GATGCAAATCATAAATTCACAGGA-3'
HRSV6：5'-TCCAGCATCTTTAAGTATCTTTATAGT-3'
Probe-HRSV9：5'-TAMRA-GGTATGTTATATGCTATGTCCAGGTTAGGAAGGG-BHQ2-3'；And
Wherein, W is A or T；
HRSV standard items sequences are：
atccgccaatcagccaaacagccaacaaaacaaccagccaatccaaaaccagccacctggaaaaaatcgacaa
tatagttacaaaaaaagaaaagggtggggcaaatatggaaacatacgtgaacaagcttcacgaaggctccacataca
cagctgctgttcaatacaatgtcctagaaaaagacgatgaccctgcatcacttacaatatgggtgcccatgttccaa
tcatctatgccagcagatttacttataaaagaactagctaatgtcaacatactagtgaaacaaatatccacacccaa
gggaccttca。
2. a kind of triple real-time fluorescence quantitative RT-PCR detection reagents of human respiratory syncytial virus according to claim 1
Box, which is characterized in that HRSV negative controls are sterile water for injection sample, and HRSV positive controls are HRSV inactivation of viruses strain samples
Product.
3. a kind of triple real-time fluorescence quantitative RT-PCR detection reagents of human respiratory syncytial virus according to claim 1
Box, which is characterized in that kit of the present invention is stored in -20 DEG C, reduces multigelation.
4. a kind of human respiratory syncytial virus according to claim 1 is universal, A types and the triple real time fluorescent quantitatives of Type B
RT-PCR detection kit, which is characterized in that the PCR reaction systems by fluorescence quantitative RT-RCR amplified reaction are in terms of 20 μ l
For：Primer HRSV1, HRSV2, HRSV3, HRSV4, HRSV5, HRSV6 final concentration are respectively 0.4 μm of ol/L, and Probe-HRSV7 is dense eventually
It spends for 0.2 μm of ol/L, the Probe-HRSV8 final concentration of 0.4 μm of ol/L of final concentration of 0.4 μm of ol/L, Probe-HRSV9,
2.5mmol/L dNTPs 2 μ L, MgCl20.8 μ L, 10 × ExTaq enzyme buffer liquid, 2 μ L, RNA reverse transcriptase, 0.4 μ L, 5U/ μ
0.4 μ L, RNA template of LExTaqDNA polymerases, 2 μ L, deionized water complement to 20 μ L of total volume.
5. a kind of human respiratory syncytial virus according to claim 1 is universal, A types and the triple real time fluorescent quantitatives of Type B
RT-PCR detection kit, which is characterized in that amplification reaction condition is：42 DEG C 5 minutes；95 DEG C 2 minutes；95 DEG C 10 seconds, 55 DEG C
40 seconds, totally 40 recycled.
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|---|---|---|---|---|
| CN113817870A (en) * | 2021-09-10 | 2021-12-21 | 宁波海尔施基因科技有限公司 | Primer composition for simultaneously detecting seven respiratory tract-related viruses and application thereof |
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| US20060003352A1 (en) * | 2004-04-29 | 2006-01-05 | Lipkin W I | Mass tag PCR for mutliplex diagnostics |
| CN104593524A (en) * | 2014-12-26 | 2015-05-06 | 江苏硕世生物科技有限公司 | Nucleic acid detection kit for rapidly detecting respiratory syncytial virus A and B and application thereof |
| CN107384958A (en) * | 2017-07-14 | 2017-11-24 | 北京交通大学 | The anti-geneome plasmids of RSV and its application based on reverse genetics structure |
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2017
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| US20060003352A1 (en) * | 2004-04-29 | 2006-01-05 | Lipkin W I | Mass tag PCR for mutliplex diagnostics |
| CN104593524A (en) * | 2014-12-26 | 2015-05-06 | 江苏硕世生物科技有限公司 | Nucleic acid detection kit for rapidly detecting respiratory syncytial virus A and B and application thereof |
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| CN113817870A (en) * | 2021-09-10 | 2021-12-21 | 宁波海尔施基因科技有限公司 | Primer composition for simultaneously detecting seven respiratory tract-related viruses and application thereof |
| CN113817870B (en) * | 2021-09-10 | 2023-12-22 | 宁波海尔施基因科技股份有限公司 | Primer composition for simultaneously detecting seven respiratory tract related viruses and application thereof |
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Application publication date: 20180615 |