CN104342498B - Culex flavivirus real-time fluorescence quantitative RT-PCR detection method and kit - Google Patents
Culex flavivirus real-time fluorescence quantitative RT-PCR detection method and kit Download PDFInfo
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Abstract
The invention discloses a kind of real-time fluorescence quantitative RT-PCR detection method of culex flavivirus and kits.The primer and TaqMan probe are designed according to the conservative region of culex flavivirus E constant gene segment C.The upstream primer for including in kit: 5'-CACGCCGAACGGACTTCT-3 ', downstream primer: 5 '-TCCATTGGCCGCCATATATC-3 ', TaqMan probe sequence: 5'-TTTCGCACCGGAGCAGCCG-3 ', 5 ' end flag F AM fluorescent reporter groups, 3 ' end label TAMRA fluorescent quenching groups, the current fragment length of amplification is 62bp.The kit can be used for detecting culex flavivirus, have good sensitivity and specificity, and the minimum virus titer of culex flavivirus can be detected for 10PFU/ reaction.
Description
Technical field
The present invention relates to molecular biology for detection viral in field of biotechnology, more particularly to culex flavivirus
Real-time fluorescence quantitative RT-PCR detection method and kit.
Background technique
Culex flavivirus (Culex flavivirus, CxFV) is a kind of mosquito biography arboviruse, belongs to flaviviridae flavivirus
Belong to, in 2003 by Japanese scholars in Culex pipiens pallens (Culex pipiens) and Culex tritaeniorhynchus (Culex
Tritaeniorhynchus virus is separated in) for the first time, then in succession in Indonesia, the U.S., West Indies Te Lini
It reaches, a variety of Culex mosquitos on the ground such as Shandong Province, Liaoning Province of African Uganda, South America Peru, Brazil, Taiwan and China
The virus is separated in worm.As for CxFV in the discovery successively of global different regions, CxFV can be divided to is two kinds of genes in recent years
There are two kinds of phenotypes of cytopathogenic effect and non-cell pathogenicity in two kinds of types in type.
Currently, the detection to CxFV in mosquito is mainly detected using the regular-PCR of virus purification and viral nucleic acid.Virus point
From time-consuming and it is not used to the non-pathogenic detection for becoming phenotype CxFV, regular-PCR is fast special, but easily causes pollution and detection sensitivity
Property is low.TaqMan Real-time PCR is a kind of detection method of real time fluorescent quantitative, passes through fluorescence intensity in detection architecture
Dynamic change to nucleic acid carry out qualitative and quantitative analysis Amplification Technologies.This research and utilization TaqMan Real-time PCR
Technology establishes molecular detecting method special for CxFV, quick, sensitive, effective, is the monitoring and detection of CxFV in medium
Provide technical support.
Summary of the invention
The present invention provides primers and TaqMan that real-time fluorescence quantitative RT-PCR detection is carried out for culex flavivirus to visit
Needle.
Primer provided by the present invention and TaqMan probe are the guarantors the most conservative according to culex flavivirus E constant gene segment C
The design of defending zone domain, it can be used to detection culex flavivirus, it can also be used to which the nucleic acid of culex flavivirus is copied in quantitative detection sample
Shellfish number.
The primer and probe sequence are respectively as follows: culex flavivirus upstream primer: CxFV-E-F:5 '-
CACGCCGAACGGACTTCT-3'；Downstream primer: CxFV-E-R:5'-TCCATTGGCCGCCATATATC-3 '；TaqMan is visited
Needle: CxFV-E-Probe:FAM5 '-TTTCgCACCggAgCAgCCg-3 ' TAMRA, 5 ' end flag F AM fluorescent reporter groups, 3 '
End label TAMRA fluorescent quenching group.
The culex flavivirus 25uL real-time fluorescence quantitative RT-PCR detects reaction system, comprising: 2 ×
ReactionMix12.5 μ L, Enzyme Mix1 μ L, 10 μM of upstream and downstream primers and each 1 μ L of 5 μM of probes, 1 μ L of RNA template,
Nuclease-free water7.5μL。
The best amplification condition of the culex flavivirus real-time fluorescence quantitative RT-PCR detection reaction system are as follows: first 45 DEG C
10min；Then 95 DEG C of 10min；Last 95 DEG C 15s and 60 DEG C 60s40 circulation.
The culex flavivirus real-time fluorescence quantitative RT-PCR detection reaction system is reacted suitable for all quantitative fluorescent PCRs
Instrument.
The viral number Quantitative Analysis Model of the culex flavivirus real-time fluorescence quantitative RT-PCR detection reaction building is minimum
Detection is limited to 10PFU/ reaction.
Culex flavivirus real-time fluorescence quantitative RT-PCR detection reaction by with regular-PCR detection method to mosquito mark
This detection application is compared, and this method has more sensitive compared with Standard PCR detection method, special, easy and is not easy dirt as the result is shown
The feature of dye.
Detailed description of the invention
Fig. 1 is the amplification curve to the specific detection of culex flavivirus；
Fig. 2 is that 10 times of real-time fluorescence quantitative RT-PCRs detection virus titer PFU for being serially diluted quantitative culex flavivirus are fixed
Amount analysis amplification curve；
Fig. 3 is the virus titer PFU quantitative analysis standard of culex flavivirus real-time fluorescence quantitative RT-PCR detection sensitivity
Curve；
Specific embodiment
Culex flavivirus real-time fluorescence quantitative RT-PCR detection method of the present invention is based on TaqMan Real-time PCR
Quantitative Real-Time TaqMan PCR Technique is become using the dynamic that the fluorescence signal accumulation generated after fluorophor is added in the reaction system
Change, thus the entire reaction process of real-time monitoring, the method that finally can carry out quantitative analysis to unknown template by standard curve.This
Invention further relates to above-mentioned detection method and establishes included all the elements.
This method specific steps are as follows:
(1) design of primer and probe
The isolated representative 50 plant library in the different regions of various years all over the world that selection GenBank is delivered
The whole genome sequence of mosquito flavivirus carries out the sequence ratio of E fragment gene with bioinformatic analysis software ClustalX1.8
It is right, according to TaqMan PCR primer, probe design principle, select the region 1031-1111 the most conservative as the purpose of amplification
Genetic fragment.Primer Express3.0 Computer Aided Design primer and probe sequence are utilized in this area, then carry out Blast
The broad spectrum activity and specificity of analysis verifying E fragment gene.Sequence information is shown in Table 1.
1 CxFV detection architecture special primer of table and probe sequence information
(2) selection of fluorescein
Fluorescent emission group selects more common FAM group, and fluorescent quenching group selects TAMRA group.
(3) synthesis of primer and probe
Primer is synthesized by Beijing Zi Xi Biotechnology Co., Ltd, and probe is synthesized by Shanghai Sheng Gong bioengineering Co., Ltd
And mark, probe 5 ' holds flag F AM fluorescent reporter group, 3 ' end label TAMRA fluorescent quenching groups.
(4) foundation of the preparation of sample and system
The strain of culex flavivirus is inoculated into aedes albopictus egg cell (C6/36 cell), obvious disease occurs in cell after 3 days
Become, collect cells and supernatant, and measuring the virus titer in culture supernatant is 1 × 106.8PFU/mL (PFU:Plaque
Forming unit), Virus culture is extracted with RNA and is carried out in BSL-2 grades of biosecurity laboratories.With QIAGEN company
QIAamp Viral RNA Mini Kit extracts viral RNA from the cells and supernatant of culex flavivirus, says referring to kit
It is bright to be operated.Using and referring to ABI company AgPath-IDTMOne-stepRT-PCRKit kit illustrates that the PCR established is anti-
Answering system is to prepare 25 μ L systems of setting concentration of component, and concrete component composition is shown in Table 2.
Table 2.PCR reaction system component constitutes table
| Component | Volume (μ L) |
| 2×Reaction Mix | 12.5 |
| Enzyme Mix | 1 |
| Upstream primer (10 μM) | 1 |
| Downstream primer (10 μM) | 1 |
| Probe (5 μM) | 1 |
| Sample rna | 2 |
| Nuclease-free water | Complement to 25 |
Reaction condition is set are as follows:
(5) verifying of culex flavivirus real-time fluorescence quantitative RT-PCR detection architecture
With Dengue 1-4 type virus, west nile virus, gene 1,3 type japanese encephalitis virus, russian spring-summer encephalitis virus, Tahyna
Virus, batai virus, getah virus, totally 3 belong to 8 kind of 11 strain virus RNA as negative control sample, exist with culex flavivirus RNA
The specificity verification of primer and probe is carried out in the reaction system of foundation.Value≤35 RNA Successful amplification Ct of culex flavivirus,
The no positive amplified signal of the RNA of his 11 negative control strains.10 times are carried out to the culex flavivirus RNA of extraction to dilute
10-1-10-44 different dilutions viral RNA, carry out 4 parallel repetition stabilities for repeating experimental verification system respectively,
The equal < 0.45 of standard deviation, the equal < 1.5% of the coefficient of variation of 4 repetition experiment gained Ct values.Specific experiment data are shown in Table 3.
Table 3.CxFV detection architecture repetition stability result
(6) in-vitro transcription of positive plasmid standard items RNA
The highly conserved region of mosquito flavivirus E fragment gene is synthesized as testing goal gene, and is cloned into PGEM-
In Teasy carrier, gene chemical synthesis and plasmid cloning are completed by Beijing Zi Xi Biotechnology Co., Ltd.With TaKaRa company
GoTaq Green Master Mix kit carries out PCR amplification to the M13 primer in cloned plasmids containing target gene, uses
QIAgen company Gel Extraction Kit kit recovery purifying PCR product, using ultraviolet nucleic acid-protein quantitative instrument to return
The linearisation DNA profiling of receipts is quantitative, with Promega company RiboMAX Large Scale RNA Production System
The RNA that T7 kit carries out target gene is transcribed in vitro.Aforesaid operations illustrate to carry out according to kit.
(7) corresponding Quantitative Analysis Model is established
The RNA that positive plasmid is transcribed in vitro is quantified, according to formula (6.02 × 1023) × (transcription product concentration ng/
μ l)/(transcription product molecular weight)=copies/ μ L calculating RNA copy number.10 times are carried out to known concentration RNA to be serially diluted
To 1 × 101~1 × 105Copies/ μ L5 sample concentration.RT-PCR expansion is carried out according to above-mentioned reaction system and reaction condition
Increase.Amplification acquired results are analyzed, according to 101~105The copy number and cycle threshold of copies/ μ L various concentration sample
(Ct) standard curve is drawn.Obtaining detection architecture calibration curve equation is Y=-3.913X+42.93, R2=0.998, amplification effect
Rate Eff=80.1%.Established culex flavivirus fluorescence quantitative RT-PCR detecting method is known most by gained standard curve
Low detection is limited to 100copies/ reaction.
To having determined virus titer (1 × 106.8PFU/mL culture supernatant) is serially diluted to obtain 1 × 101~1
×106PFU/mL totally 7 gradients extract each dilution cell with the QIAamp Viral RNA Mini Kit of QIAGEN company
Viral RNA in culture supernatant illustrates to be operated referring to kit.It is carried out according to above-mentioned established system and reaction condition
RT-PCR amplification.According to 1 × 101~1 × 106The copy number and cycle threshold (Ct) of PFU/mL various concentration sample draw standard
Curve.Obtaining detection architecture calibration curve equation is Y=-3.551X+37.56, R2=0.994, amplification efficiency Eff=
91.3%.The lowest detection for the culex flavivirus fluorescence quantitative RT-PCR detecting method established is limited to 10PFU/mL.
(8) application in practical Mosquito specimen is compared with
Simultaneously 246 batches of Mosquito specimens are carried out with the viral core of CxFV with two methods of regular-PCR method and TaqMan PCR
Acid detection, 9 parts of nucleic acid positive sample of regular-PCR detection, positive 16 parts of (the Ct < 35) sample of TaqManPCR detection nucleic acid (including
Regular-PCR is 9 parts positive).Show that this method has higher sensibility.
(9) result judges
The positive is judged to when Ct (threshold value)≤35 of sample to be examined；It need to be examined again as 35 < Ct < 40 of sample to be examined
It surveys, is judged to the positive if result is still between 35 < Ct < 40, is otherwise judged to feminine gender.The sample to be examined of no Ct value is judged to feminine gender.
The invention has the following advantages that
1. establishing culex flavivirus real-time fluorescence quantitative RT-PCR detection method for the first time, detection method is used directly for
Any sample extracts the culex flavivirus quantitative detection after nucleic acid.
2. detection method is compared with other culex jaundice virus detection methods, sensitivity is higher, and the operation is more convenient and can
It effectively prevent polluting, testing result is more intuitive accurate, and detection efficiency is made to be greatly improved.
3. detection method can not only evaluate the infection state of mosquito flavivirus in sample, at the same also for the virus with
Further research in terms of disease relationship provides effective tool.
In conclusion the object of the invention is to be established using TaqMan Real-time round pcr specifically for library
Special, quick, more sensitive, the effective molecular detecting method of mosquito flavivirus.Culex flavivirus is picked out by sequence alignment
Sequence highly conserved in E genetic fragment in genome designs pair of primers and a TaqMan probe in this sequence, establishes
Real-time fluorescence quantitative RT-PCR reaction system, the specificity and stability for carrying out system are verified, and Preliminary Applications are in practical mosquito
The detection of sample.The method of the present invention mentions for the detection of culex flavivirus in mosquito medium from now on and the research of the virus related fields
Effective tool is supplied.
Claims (1)
1. a kind of culex flavivirus real-time fluorescence quantitative PCR detection kit, which is characterized in that the kit includes a pair of special
Specific primer and a specificity fluorescent probe, primer and probe are the guarantors the most conservative according to culex flavivirus E constant gene segment C
The design of defending zone domain, expand the nucleotide sequence of target are as follows: 5 '-CACGCCGAACGGACTTCTTGAGTTTCGCACCGGAGCA
GCCGAGATATATGGCGGCCAATGGA-3 ', amplification target fragment length are 62bp, and it is yellow to be used for quantitatively detecting culex in sample
The nucleic acid copies of virus；The primer is respectively as follows: with probe sequence
Primer: CxFV-E-F:5 '-CACGCCGAACGGACTTCT-3 '
CxFV-E-R:5’-TCCATTGGCCGCCATATATC-3’
Probe: CxFV-E-Probe:FAM 5 '-TTTCgCACCggAgCAgCCg-3 ' TAMRA,
Wherein, the FAM of the end of probe 5 ' label is fluorescent reporter group, and the TAMRA of 3 ' end labels is fluorescent quenching group.
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