CN104017901B - A kind of method and test kit simultaneously detecting people A type, Type B respiratory syncytial virus and human metapneumovirus - Google Patents
A kind of method and test kit simultaneously detecting people A type, Type B respiratory syncytial virus and human metapneumovirus Download PDFInfo
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Abstract
The invention belongs to biological technology application, relate to a kind of the multi-fluorescence RT-PCR method containing interior Quality Control and the test kit that detect people A type, Type B respiratory syncytial virus and human metapneumovirus simultaneously.The present invention is directed to conserved sequence design Auele Specific Primer and the probe of the N gene of people A type, Type B respiratory syncytial virus and human metapneumovirus representative strains, L gene and L gene, establish the single stage method multi-fluorescence RT-PCR method for quick containing interior Quality Control, simple and efficient to handle, overcome the loaded down with trivial details of conventional substance fluorescence RT-PCR single hole list inspection, simplify schedule of operation, save experimentation cost, the aspects such as the Site Detection that the specificity utilizing it higher, sensitivity, high efficiency and stability are virus, evaluation of hygiene, clinical diagnosis provide strong technical support.
Description
Technical field
The present invention relates to biological technology application, in particular for Detection and Identification while people A type, Type B respiratory syncytial virus and human metapneumovirus.
Background technology
Human respiratory syncytial virus (Humanrespiratorysyncytialvirus, hRSV) causes one of infant, oldaged physically weak person, the most common, the most important pathogenic agent of immunocompromised person's winter-spring season lower respiratory infection.RSV belongs to Paramyxoviridae pneumonitis virus and belongs to, genome is sub-thread strand RNA, be made up of 15222 Nucleotide, to encode 11 kinds of protein, wherein 3 is cross-film envelope glycoprotein F protein (fusionproteion), G-protein (attachmentprotein) and SH albumen (smallhydrophobicprotein), according to the antigenic difference of G-protein, RSV is divided into A, B amphitypy, China popular based on A type RSV.
Human metapneumovirus (humanmetapneumovirus, hMPV) be single minus-stranded rna virus, belong to Paramyxoviridae Pneumovirinae, it is a kind of new respiratory pathogen first found by Dutch scholar calendar year 2001, current global distribution, the patient that hMPV infects shows from upper respiratory tract infection symptoms to acute bronchitis and pneumonia, and sings and symptoms mainly comprises cough, pharyngalgia, runny nose and heating etc., and asthma, expiratory dyspnea etc. can appear in severe patient.HMPV particle is polymorphism, and genome is about 13kb, and the sequence of hMPV gene 3 ' ~ 5 ' is N-P-MF-M2-SH-G-L, and wherein N genes encoding nucleocapsid rna binding protein, relatively conservative, becomes the target area of molecular Biological Detection.
At present, the classical way detecting common Respirovirus is both at home and abroad the separation and Culture (WHO recommendation) of virus, but its operation is loaded down with trivial details, and expend time in length, Electronic Speculum, immunohistochemical methods, ELISA, Standard PCR etc. have advantage outstanding separately, but be all difficult to detect trace dna and accurate quantitative analysis, the Real-Time Fluorescent Quantitative PCR Technique (Real-timefluorescentquantitativePCR) that 20 end of the centurys grew up is that one adds fluorophor in PCR reaction system, fluorescent signal is utilized to accumulate the whole PCR process of Real-Time Monitoring, this technology not only achieves the quantitative and qualitative analysis to template, and have highly sensitive, specificity and reliability stronger, multiple reaction can be tested, level of automation is high, nonstaining property, there is the feature such as real-time and accuracy.Thus, in respiratory pathogen detects, there is huge applications advantage.Relative regular-PCR and single fluorescent PCR, multiple fluorescence PCR has the following advantages: (1) detection efficiency is high, namely multiple fluorescence PCR can realize a pipe and examines more, distinguishes different detected objects by the fluorescent signal of the different wave length collected, thus the flow process that simplifies the operation; (2) saving reagent cost and human users's time, especially when carrying out a large amount of sample detection, testing cost and operating time can be reduced significantly.(3) monitoring of interior Quality Control gene is contained in present method, in all kinds of sample, there is the factor of various impact or suppression PCR reaction, and in the acquisition process of sample, nucleic acid extraction, amplification and analytic process, also may cause because of human operational error producing false negative detected result.Therefore the application adopts the method for interior Quality Control, and in containing in sample, Quality Control gene (LDHA) experience detects the whole process of sample nucleic acid extraction, application of sample, pcr amplification and signal detection, thus can to every a sample implementing monitoring.
What the present invention successfully set up can detect people A type, Type B respiratory syncytial virus and human metapneumovirus single stage method multi-fluorescence RT-PCR method simultaneously, adopt LDHA as interior Quality Control, can PCR restraining factors in effective monitoring sample and the false negative result that caused by operate miss.And the method except having high specificity, highly sensitive, detection efficiency is high and except the feature of stability, it is less that it detects required RNA amount, and fast easy and simple to handle, in the Site Detection, evaluation of hygiene, clinical diagnosis etc. of virus, demonstrate good application prospect.And be conducive to furtheing investigate further related diseases pathogen infection molecule mechanism and immune adjusting mechanism, carry out the aspects such as effective clinical treatment and demonstrate good application prospect.
Summary of the invention
The present invention is supported by country " 12 " scientific and technological key special subjects 2012ZX10004206-002.Our successful design and having synthesized for the corresponding N gene of people A type, Type B respiratory syncytial virus and human metapneumovirus, L gene and L gene-specific primer and probe.Based on this primer and probe, we establish the multi-fluorescence RT-PCR detection method of single stage method containing interior Quality Control.The method by comparing with commercialization substance fluorescence RT-PCR diagnostic kit the detection of various respiratory road virus, the structure of typical curve, replica test etc., illustrate that the method has higher specificity, sensitivity, high efficiency and stability.
In a first aspect of the present invention, provide a kind of Auele Specific Primer for people A type, Type B respiratory syncytial virus and human metapneumovirus N gene, L gene and L gene and probe sequence, as shown in SEQNO:1 to 12.
In a second aspect of the present invention, provide a kind of test kit simultaneously detecting people A type, Type B respiratory syncytial virus and human metapneumovirus, containing primer and probe described in first aspect in this test kit.
In one embodiment, described test kit comprises following composition:
A) reaction buffer, described damping fluid is Standard PCR reaction buffer;
B) reaction enzymes, described reaction enzymes is Standard PCR reaction enzymes;
C) SEQNO:1,2,4,5,7,8, the primer shown in 10,11;
D) SEQNO:3,6, the probe shown in 9,12
E) without the water of RNA enzyme.
It is characterized in that the N gene probe 5 ' end of the people A type respiratory syncytial virus as shown in SEQNO:3 is marked by FAM, 3 ' end is marked by BHQ1; The L gene probe 5 ' end of the human B-type respiratory syncytial virus as shown in SEQNO:6 is marked by JOE, and 3 ' end is marked by BHQ1; The L gene probe 5 ' end of the human metapneumovirus as shown in SEQNO:9 is marked by ROX, and 3 ' end is marked by BHQ2; In as shown in SEQNO:12, Quality Control gene LDHA gene probe 5 ' end is marked by CY5, and 3 ' end is marked by BHQ2.
In a specific embodiment, described method is the embody rule of multi-fluorescence RT-PCR detection kit, and uses described primer and probe as detection sequence.
In a third aspect of the present invention, provide a kind of PCR amplification method simultaneously detecting people A type, Type B respiratory syncytial virus and human metapneumovirus, the method uses the test kit described in second aspect.
In one embodiment, said method comprising the steps of:
A) setting of four fluorescence channels;
B) temperature and time of reverse transcription;
C) temperature and time of preheating;
D) thermal cycling (preheating temperature and time, annealing temperature and time, phosphor collection temperature, cycle number);
E) with the Ct value defined to judge detected result.
In a fourth aspect of the present invention, provide the application in the detection reagent of preparation people A type, Type B respiratory syncytial virus and human metapneumovirus of primer described in first aspect and probe.
Accompanying drawing explanation
Fig. 1. people A type, Type B respiratory syncytial virus and human metapneumovirus single stage method contain the specificity identification of the multi-fluorescence RT-PCR detection method of interior Quality Control, wherein:
Figure 1A is the amplified fluorescence figure simultaneously adding people A type, Type B respiratory syncytial virus and human metapneumovirus positive template.Figure 1B is the amplified fluorescence figure (for Influenza B virus) of the positive template adding other 7 kinds of many cause of diseases of respiratory tract.
Embodiment
People A type of the present invention, Type B respiratory syncytial virus and human metapneumovirus single stage method multi-fluorescence RT-PCR detection method are the Auele Specific Primer based on corresponding type N gene, L gene and L gene conserved regions design and probe.
Technological approaches of the present invention be first utilize AppliedBiosystems (ABI) company to develop PrimerExpress3.0 software, for the conserved regions design Auele Specific Primer of the corresponding N gene of people A type, Type B respiratory syncytial virus and human metapneumovirus, L gene and L gene and probe sequence, determine best preparation system and upper machine amplification program, and carry out the confirmatory experiments such as specificity, sensitivity and stability, successfully establish people A type, Type B respiratory syncytial virus and human metapneumovirus single stage method multi-fluorescence RT-PCR method for quick.
The present invention is set forth further below in conjunction with preferred embodiment.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.Unreceipted concrete experimental technique in the following example, usual conveniently condition and method, as Molecular Cloning: A Laboratory room handbook (Sambrook, etal.NewYork:ColdSpringHarborLaboratoryPress, 1989) and the method that described in real-time fluorescence PCR technology (Li Jinming, 2007), method or reagent manufacturers provide.
Simultaneously, it is to be noted, detection method of the present invention and test kit are not only applied to and detect the sample of patient (people, animal), also comprise the detection of the non-diagnostic object to several samples such as the water sample in environment, foodstuff samples, air sample, microbiological specimens, zooblast samples.Its application is also all the common technology means of art technology, does not need to carry out special, creative change.
Embodiment 1. people A type, Type B respiratory syncytial virus and each hypotype N gene of human metapneumovirus, L gene and L gene-specific primer and probe design and synthesis
The corresponding target genes N gene of 1.1 people A types, Type B respiratory syncytial virus and human metapneumovirus representative strains, L gene and the selection of L gene and the determination of conserved regions
The sequence of this institute is selected from NCBIGenBank (http://www.ncbi.nlm.nih.gov), and main selection gist is: (a) age nearlyer strain; B () has full length sequence and main body sequence strain; C () chooses representative strain with after subtype sequences comparison.The information of the corresponding N gene of people A type, Type B respiratory syncytial virus and the human metapneumovirus representative strains chosen, L gene and L gene is in Table 1-1,1-2 and 1-3.
The corresponding N gene of selected people A type, Type B respiratory syncytial virus and human metapneumovirus representative strains, L gene and L gene are input in DNAssist software by this research respectively carries out homology comparison, determine the sequence conservation of corresponding N gene, L gene and L gene, for primer and probe design provide target area.
Table 1-1. chooses the N gene information that people A type respiratory syncytial virus (hRSVA) represents strain
Table 1-2. chooses the L gene information that human B-type respiratory syncytial virus (hRSVB) represents strain
Table 1-3. chooses the L gene information that human metapneumovirus (hMPV) represents strain
The Design and synthesis of 1.2 primers and probe
Primer and probe be utilize AppliedBiosystems (ABI) company to research and develop PrimerExprcss3.0 software, for the conserved regions design Auele Specific Primer of the corresponding N gene of people A type, Type B respiratory syncytial virus and human metapneumovirus, L gene and L gene and TaqMan probe sequence, and primer and probe mass are assessed, selected detection people A type, Type B respiratory syncytial virus and the Auele Specific Primer of human metapneumovirus and the primer of probe sequence and interior Quality Control gene LDHA and probe sequence are synthesized by precious biological (Dalian) company of TakaRa.The N gene probe 5 ' end of the people A type respiratory syncytial virus wherein as shown in SEQNO:3 is marked by FAM, and 3 ' end is marked by BHQ1; The L gene probe 5 ' end of the human B-type respiratory syncytial virus as shown in SEQNO:6 is marked by JOE, and 3 ' end is marked by BHQ1; The L gene probe 5 ' end of the human metapneumovirus as shown in SEQNO:9 is marked by ROX, and 3 ' end is marked by BHQ2; In as shown in SEQNO:12, Quality Control gene LDHA gene probe 5 ' end is marked by CY5, and 3 ' end is marked by BHQ2.In order to reduce the interference of reaction system, the primer of synthesis and probe adopt HPLC to carry out purifying (see table 2).
Table 2. is selected detects people A type respiratory syncytial virus (hRSVA), the primer of human B-type respiratory syncytial virus (hRSVB) and human metapneumovirus (hMPV) and interior Quality Control gene LDHA and probe sequence and labelling groups
The foundation of embodiment 2. people A type, Type B respiratory syncytial virus and human metapneumovirus single stage method multi-fluorescence RT-PCR detection system
2.1 according to hero company nucleic acid extraction kit (
viralRNAMiniKit) sample nucleic acid is extracted.
The preparation reagent preparation of 2.2 people A types, Type B respiratory syncytial virus and human metapneumovirus single stage method multi-fluorescence RT-PCR detection system adopts the AgPath-IDTMOne-stepRT-PCRKit of Ambion company, and system is 50 μ l:
2×RT-PCRbuffer25μl
25×RT-PCREnzyme2μl
DetectionEnhancer3μl
Primer, probe add each 0.5 μ 1 of primer as shown in SEQNO:1,2,4,5,7,8,10,11 simultaneously, and primer final concentration is 300nM; Add each 0.5 μ l of probe primer as shown in SEQNO:3,6,9,12, final concentration is 150nM
(three kinds of positive templates respectively add 2 μ l to template 6 μ l, and often kind of template is to add after 10-1 multiple dilutions, final concentration 1pg-100ng.Using the positive nucleic acid of respiratory syncytial virus as negative control template)
Water without RNA enzyme supplies system to 50 μ l
The upper machine amplification program of 2.3 people A types, Type B respiratory syncytial virus and human metapneumovirus single stage method multi-fluorescence RT-PCR detection method
Adopt the Roche480 instrument of Roche Holding Ag to carry out upper machine amplification, its program is as follows:
FAM, JOE, ROX, CY5 tetra-fluorescent collecting passages are first set;
Reverse transcription temperature and time: 45 DEG C, 20min;
Denaturation temperature and time: 95 DEG C, 10min;
Result criterion is: Ct value≤34, are judged to be the positive; Ct value >=36, are judged as feminine gender; Ct value is suspicious between 34 ~ 36.
The specificity identification of embodiment 3. people A type, Type B respiratory syncytial virus and human metapneumovirus single stage method multi-fluorescence RT-PCR detection method
The multi-fluorescence RT-PCR reaction system utilizing embodiment 2 to set up is respectively to people A type respiratory syncytial virus, human B-type respiratory syncytial virus, human metapneumovirus, influenza A virus, Influenza B virus, influenza virus C, parainfluenza virus, enterovirus, adenovirus, the positive nucleic acid of bocavirus detects, its result shows, only has people A type, Type B respiratory syncytial virus and human metapneumovirus are respectively at FAM, JOE, there is corresponding specificity fluorescent amplification curve in ROX sense channel, and not there is cross reaction, and other 7 strain virus have amplification curve except interior Quality Control CY5 passage, other three passages are then without amplification curve, illustrate that the method has very strong specificity (see Fig. 1).
The sensitivity qualification of embodiment 4. people A type, Type B respiratory syncytial virus and human metapneumovirus single stage method multi-fluorescence RT-PCR detection method
The detection limit of 4.1 single stage method multi-fluorescence RT-PCR detection method
With for people A type respiratory syncytial virus, human B-type respiratory syncytial virus and human metapneumovirus, respective N gene, L gene and L gene-specific primer, carry out pcr amplification (its amplification scope comprises fluorescence quantitative RT-RCR amplification region), its product utilization XhoI and HindIII (NewEnglandBiolabs company) is connected on pcDNAII carrier (Invitrogen company), clone again, extract plasmid, called after pcDNAII-hRSVA respectively, pcDNAII-hRSVB, pcDNAII-hMPV, and plasmid is sent precious biological (Dalian) company order-checking confirmation, the correct plasmid of order-checking is used for the typical curve setting up multi-fluorescence RT-PCR detection method.PcDNAII-hRSVA, pcDNAII-hRSVB, pcDNAII-hMPV positive plasmid trace dna quantitative instrument NanoDrop (model: ND-1000) carries out quantitatively, get the recombinant plasmid of 101,102,103,104,105,106,107,108 copy/μ l as standard substance template, join respectively in the single stage method multi-fluorescence RT-PCR reaction system of embodiment 2 foundation and increase, the Log value according to Ct value and template concentrations builds typical curve.Result shows, Ct value and the template concentrations Log value of the typical curve built by pcDNAII-hRSVA positive plasmid have good linear relationship in (101 ~ 106), relation conefficient is 0.9978, according to the criterion of multi-fluorescence RT-PCR detection method result, detection limit is 20 copies.Ct value and the template concentrations Log value of the typical curve built by pcDNAII-hRSVB positive plasmid have good linear relationship in (101 ~ 106), relation conefficient is 0.9895, according to the criterion of multi-fluorescence RT-PCR detection method result, detection limit 40 copies.Cycle threshold (Ct value) and the template concentrations Log value of the typical curve built by pcDNAII-hMPV positive plasmid have good linear relationship in (101 ~ 106), relation conefficient is 0.9991, according to the criterion of multi-fluorescence RT-PCR detection method result, detection limit is 40 copies.Illustrate thus, single stage method multi-fluorescence RT-PCR detection method has higher sensitivity.
4.2 single stage method multi-fluorescence RT-PCR compare with conventional RT-PCR detection sensitivity
The positive nucleic acid of people A type, Type B respiratory syncytial virus and human metapneumovirus carries out 10 times of gradient series dilution (103 ~ 10-8), the multi-fluorescence RT-PCR method set up by embodiment 2 and conventional RT-PCR method detect it, and the concrete steps of conventional RT-PCR method are as follows: according to SuperScriptTMIlIOne-StepRT-PCRSystemwith
taqDNAPolymerase (Cat.No.12574-018) specification sheets carries out system preparation: 2 × ReactionMix is 25 μ L, and upstream primer is 1 μ L, and downstream primer is 1 μ L, SuperScriptTMIII
taqMix is 2 μ L, and positive nucleus acid template 1 μ L is 20 μ L without RNA enzyme water, adds up to 50 μ L systems.The coded program of PCR instrument (U.S. Biorad, model T100): reverse transcription 50 DEG C, 30min, denaturation 94 DEG C, 2min, (preheating 94 DEG C, 15s anneal 58 DEG C, 30s in pcr amplification 40 circulation, increase 68 DEG C, 1min), finally extend 68 DEG C, 5min.Pcr amplification product is splined on 1% sepharose, electrophoresis, qualification target clip size determines detected result.By the synchronous detection to the different extension rate of people A type, Type B respiratory syncytial virus and human metapneumovirus positive template, its result shows, multi-fluorescence RT-PCR method improves 100 times (see table 3) than conventional RT-PCR method detection sensitivity.
Table 3. single stage method multi-fluorescence RT-PCR method compares with conventional RT-PCR method detection sensitivity
" * " represents that detected result is positive; " # " represents that detected result is negative.
The qualification of embodiment 5. people A type, Type B respiratory syncytial virus and human metapneumovirus single stage method multi-fluorescence RT-PCR method detection efficiency
The multi-fluorescence RT-PCR method utilizing embodiment 2 to set up detects great amount of samples, and compares with the substance fluorescence RT-PCR method of the Zhijiang River, Shanghai company man A type respiratory syncytial virus nucleic acid detection kit, human B-type respiratory syncytial virus nucleic acid detection kit, human metapneumovirus nucleic acid detection kit.Result shows, multi-fluorescence RT-PCR compares with Zhijiang River test kit that its susceptibility is 97.74%, specificity 100%, consistence 99.8% (see table 4).In this research, the LDHA in whole sample detects and is the positive, shows the appearance not causing false negative result in testing process because of misoperation or the RT-PCR response inhabitation factor.At present, it is less that more ripe single stage method multi-fluorescence RT-PCR method is set up, and substance fluorescent RT-PCR method for detecting is widely used, when its sensitivity, specificity and consistence are suitable, multi-fluorescence RT-PCR method achieves pipe four inspection, simplify procedures, consuming time is 1/3 of substance fluorescence RT-PCR, reach save time, laborsaving, the effect of economizing nucleic acid, cost-saving, have more high efficiency and flux properties, when especially carrying out great amount of samples screening, its advantage is more obvious.
Table 4. single stage method multi-fluorescence RT-PCR compares with substance fluorescence RT-PCR (commercial test kit) detected result
The repeated pruning of embodiment 6. people A type, Type B respiratory syncytial virus and human metapneumovirus single stage method multi-fluorescence RT-PCR detection method
In same single test (same PCR plate), detect the multi-fluorescence RT-PCR method that the positive nucleic acid of the people A type of 10 times of gradient dilutions, Type B respiratory syncytial virus and human metapneumovirus (arrange 10-1,10-2,10-3 and dilute gradient) utilizes embodiment 2 to set up, each sample does 3 repetitions; Repeat 3 times in different PCR plate in addition, the variation coefficient of batch Ct value that interior three reaction repeated obtain of each extent of dilution sample of result is between (0.26% ~ 0.59%), the variation coefficient repeated between batch is between (0.28% ~ 0.83%), show that the multi-fluorescence RT-PCR detection method error that the present invention sets up is little, reproducible, can people A type, Type B respiratory syncytial virus and human metapneumovirus be stablized, be detected reliably.
Claims (2)
1. detect a test kit for people A type, Type B respiratory syncytial virus and human metapneumovirus simultaneously, it is characterized in that its sequence is respectively containing Auele Specific Primer and probe in described test kit:
1) SEQNO:1,2,4,5,7,8, the primer shown in 10,11;
2) SEQNO:3,6, the probe shown in 9,12.
2. the application of test kit according to claim 1 in the diagnostic reagent of preparation people A type, Type B respiratory syncytial virus and human metapneumovirus.
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| CN104450974B (en) * | 2014-12-30 | 2017-09-22 | 重庆医科大学附属儿童医院 | A kind of hMPV quantitative typings detection kit and its detection method |
| CN105543414B (en) * | 2016-01-22 | 2019-05-21 | 广州医科大学附属第一医院 | A kind of Respiratory Syncytial Virus(RSV) A/B hypotype multiple fluorescence quantitative PCR detection primer group and probe groups and its kit and preparation method |
| CN108411039B (en) * | 2018-05-14 | 2021-06-11 | 南京岚煜生物科技有限公司 | Kit for detecting respiratory syncytial virus A, B type based on microfluidic chip and use method thereof |
| CN108384898B (en) * | 2018-05-14 | 2021-04-13 | 南京岚煜生物科技有限公司 | Kit for detecting respiratory syncytial virus A, B type and use method thereof |
| CN109762942B (en) * | 2019-03-13 | 2022-05-17 | 中国疾病预防控制中心病毒病预防控制所 | Internal reference-containing double isothermal nucleic acid amplification method for rapidly detecting respiratory syncytial virus |
| CN114292958A (en) * | 2021-12-28 | 2022-04-08 | 无锡科智达科技有限公司 | Respiratory tract pathogen multi-connection detection kit and application thereof |
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| CN101107366A (en) * | 2004-12-24 | 2008-01-16 | 卡洛斯Ⅲ世健康研究所 | Probes and methods for the simultaneous detection and identification of multiple viruses that cause respiratory infections in humans |
| CN101985665A (en) * | 2010-11-12 | 2011-03-16 | 复旦大学 | Method for detecting various respiratory viruses and primers and probes thereof |
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