CN102312017A - Real-time reverse transcription-polymerase chain reaction (RT-PCR) detection method and kit for Coxsackie virus - Google Patents
Real-time reverse transcription-polymerase chain reaction (RT-PCR) detection method and kit for Coxsackie virus Download PDFInfo
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Abstract
The invention relates to a real-time reverse transcription-polymerase chain reaction (RT-PCR) detection method and a kit for a Coxsackie virus. Specifically, the invention discloses a method for detecting the Coxsackie virus, which is characterized in that a polymerase chain reaction is carried out in a polymerase reaction system. The polymerase reaction system comprises an amplification Coxsackie virus-contained specific primer pair and a Coxsackie virus specific probe. The invention also provides the corresponding kit. The invention is capable of sensitively, simply and conveniently detecting and verifying the Coxsackie virus.
Description
Technical field
The present invention relates to molecular biology and nucleic acid detection technique field.Particularly, the present invention relates to be directed against real-time fluorescent RT-PCR method for detecting and the test kit that Coxsackie virus detects.
Background technology
Coxsackie virus (Coxsackievirus) belongs to pico+ribonucleic acid+virus section (Picornaviridae) enterovirus genus, is divided into two types according to its biology characteristics, i.e. A group and B group.Cause herpangina (A group) and non-paralytic PLD (B group) after human infection's Coxsackie virus respectively.According to investigations in the acute calenture with pars oralis pharyngis bleb and fash, 79% is due to the Ke Sake virus of A group, and wherein more common coxsackie virus A 16-type is one of main pathogens that causes hand foot mouth disease.The CB group is to cause the myocarditic main pathogenic microbes of human virus's property
[1]
In recent years, in Beijing, a lot of Coxsackie virus infection incidents successively take place in ground such as Henan, Shenyang
[2-4]In May, 2005, CB 5 type epidemic situations together took place in Henan, lasted 34 days morbidity 49 examples, and the attack rate of children below 14 years old reaches 13.57%
[2]China hygiene department statistics in 2006, all parts of the country report that altogether hand foot mouth disease reaches 13637 examples
[4], it is to infect due to the A16 type Coxsackie virus that quite a few case is arranged.
Do not develop at present the vaccine of effective prevention Coxsackie virus as yet, it is particularly important therefore to strengthen the Coxsackie virus prevention and control.In order to find the virus infection source timely and effectively, setting up efficiently, the Coxsackie virus detection method is one of prophylaxis of viral infections and popular gordian technique means.
Yet, in Coxsackie virus detects, have many difficult points at present.Viral level is very low in the environmental sample, generally at 100 below the virion, and disturbs and to suppress the factor of subsequent detection complicated more, has brought huge challenge to detection method, therefore needs the stronger and higher detection method of sensitivity of specific aim.The investigator had reported the methods such as ELISA method, regular-PCR and real-time fluorescence PCR of Coxsackie virus in recent years in succession both at home and abroad
[5-8]Though the ELISA method can detect the IgM and the IgG antibody of Coxsackie virus among the early stage patients serum, this method is restricted application because of Antibody Preparation is difficult, detection sensitivity is not high.
PCR detection technique cost based on nucleic acid is low and quick, has been widely used in virus and has detected.Former study is many based on the Taqman-FAM-TAMRA probe, for example: Tana (2008) etc. sets up the multiple real time fluorescence PCR method of coxsackie virus A 16-type and enterovirns type 71
[5]Yet,, what this method adopted when detecting the A16 C-type virus C is the fluorescent hybridization probe, and it has had higher requirement to the real-time fluorescence PCR system, and not all real-time fluorescence PCR appearance all can be implemented reaction.The fluorescent hybridization probe needs 2 adjacent probes, has increased cost like this, and wherein a probe mark LC Red 750 fluorophors, this group resolving power is limited, poor sensitivity.Therefore, this method is unfavorable for applying.
This area still lacks the strong detection method of highly sensitive, high specific, flexible and convenient, applicability to single serotype Coxsackie virus, more lacks rapid detection and the detection technique of identifying various serotype Coxsackie viruss.
In sum, in view of still there not being at present good, the highly sensitive Coxsackie virus real-time fluorescent RT-PCR method for detecting of specificity.Therefore, this area presses for good, the highly sensitive Coxsackie virus real-time fluorescent RT-PCR method for detecting of exploitation specificity.
Summary of the invention
The object of the present invention is to provide good, highly sensitive, accurate, the easy Coxsackie virus real-time fluorescent RT-PCR method for detecting of a specific specificity.
Another object of the present invention is to provide Coxsackie virus real-time fluorescent RT-PCR detection reagent box.
A purpose more of the present invention is to provide Coxsackie virus to detect relevant primer and probe.
In first aspect of the present invention; A kind of polymerase chain reaction method is provided; It comprises step: in the polymeric enzyme reaction system, carry out the polymerase chain reaction; The Auele Specific Primer that contains the Coxsackie virus that increases in the described reaction system to the Coxsackie virus specific probe, and the amplified production that goes out of said primer amplification has the nucleotide sequence in Coxsackie virus genome VP1 district.
In another preference, the Auele Specific Primer of described amplification Coxsackie virus is right to comprising the 1-5 kind primer that is selected from down group:
A9 type Coxsackie virus: SEQ ID NO:1 and SEQ ID NO:2;
A16 type Coxsackie virus: SEQ ID NO:4 and SEQ ID NO:5;
B2 type Coxsackie virus: SEQ ID NO:7 and SEQ ID NO:8;
B3 type Coxsackie virus: SEQ ID NO:10 and SEQ ID NO:11;
B5 type Coxsackie virus: SEQ ID NO:13 and SEQ ID NO:14.
In another preference, described Coxsackie virus specific probe is Taqman-MGB probe or Taqman probe.
In another preference, the copy number of Coxsackie virus genome or cDNA preferably is less than or equal to 5 less than 10 in the described polymeric enzyme reaction system, more preferably is less than or equal to 2.
In another preference, described Coxsackie virus specific probe has the nucleotide sequence of the group of being selected from down:
A9 type Coxsackie virus: SEQ ID NO:3;
A16 type Coxsackie virus: SEQ ID NO:6;
B2 type Coxsackie virus: SEQ ID NO:9;
B3 type Coxsackie virus: SEQ ID NO:12;
B5 type Coxsackie virus: SEQ ID NO:15.
In another preference, said method also comprises step: in the process of polymerase chain reaction or afterwards, detect the fluorescent signal that the Coxsackie virus specific probe sends.
In another preference, contain nucleic acid in the described reaction system from environmental sample to be detected or human excrement sample.
In second aspect of the present invention, a kind of detection kit is provided, it contains following reagent:
(a) Auele Specific Primer of amplification Coxsackie virus is right, and the amplified production that goes out of said primer amplification has the nucleotide sequence in Coxsackie virus genome VP1 district; With
(b) the detection specificity probe of detection Coxsackie virus.
In another preference, the Auele Specific Primer of described amplification Coxsackie virus is right to comprising the 1-5 kind primer that is selected from down group:
A9 type Coxsackie virus: SEQ ID NO:1 and SEQ ID NO:2;
A16 type Coxsackie virus: SEQ ID NO:4 and SEQ ID NO:5;
B2 type Coxsackie virus: SEQ ID NO:7 and SEQID NO:8;
B3 type Coxsackie virus: SEQ ID NO:10 and SEQ ID NO:11;
B5 type Coxsackie virus: SEQ ID NO:13 and SEQ ID NO:14.
In another preference, described Coxsackie virus specific probe is Taqman-MGB probe or Taqman probe.
In another preference, described Coxsackie virus specific probe has the nucleotide sequence of the group of being selected from down:
A9 type Coxsackie virus: SEQ ID NO:3;
A16 type Coxsackie virus: SEQ ID NO:6;
B2 type Coxsackie virus: SEQ ID NO:9;
B3 type Coxsackie virus: SEQ ID NO:12;
B5 type Coxsackie virus: SEQ ID NO:15.
In another preference, a kind of detection kit that is used to detect Coxsackie virus is provided, it contains:
(a) to 5 pairs of Auele Specific Primers of 5 kinds of serotypes of Coxsackie virus (A9 type, A16 type, B2 type, B3 type, B5 type) design, described Auele Specific Primer is to one section sequence in five kinds of serotype genomes of Coxsackie virus VP1 district that increases specifically respectively;
(b) Taqman-MGB probe, described Taqman-MGB probe combines with corresponding primer amplified regional DNA specificity;
(c) marker of choosing wantonly;
In another preference, described marker is Coxsackie virus cDNA or contains the segmental DNA of Coxsackie virus amplification purpose;
In another preference, described Auele Specific Primer and/or probe are selected from down group:
Wherein, S:C/G; M:A/C; W:A/T; Y:T/C; V:A/C/G; R:A/G; D:A/G/T; K:G/T.
In the third aspect of the invention, the purposes of the described test kit of second aspect present invention is provided, be used for detecting whether there is Coxsackie virus at environmental sample or human excrement sample.
In another preference, described environmental sample comprises: instant food such as fishery products such as water source, shellfish, fruits and vegetables, salad etc.; Described human excrement sample comprises vomitus, movement of the patient that falls ill etc.
In fourth aspect of the present invention, a series of oligonucleotide fragments are provided, they have the nucleotide sequence that is selected from SEQ IDNO.1-15.Described oligonucleotide can be used as primer or probe.
Aspect the of the present invention the 5th, a series of oligonucleotide fragments are provided or have contained said segmental plasmid, they have the nucleotide sequence that is selected from SEQ ID NO.16-20.Described oligonucleotide fragment or plasmid can be used as marker.
Aspect the of the present invention the 6th; The Auele Specific Primer that the above-mentioned specific amplification Coxsackie virus of the present invention is provided is to the purposes of, Coxsackie virus specific probe and/or marker, and they are used to prepare and are used for detecting the test kit that whether has Coxsackie virus at sample.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and specifically described each technical characterictic can mutual combination in (like embodiment) hereinafter, thus constitute new or optimized technical scheme.As space is limited, this tired no longer one by one stating.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Figure 1A-1E has shown the amplification curve and the typical curve of five kinds of serotype Coxsackie virus real-time fluorescence RT-PCR detection architecture.Each figure is respectively (A) pMD-B2; (B) pMD-B3; (C) pMD-B5; (D) pMD-A9; (E) pMD-A16.Be template with 10 times of gradient dilution liquid of plasmid control molecule wherein, figure upper curve template amount from left to right is respectively 2.0E+6 to 2.0E+1 copy plasmid control molecule DNA.Wherein shown between Ct value and the template copy number and had good linear relationship.
Fig. 2 has shown the marker nucleotide sequence of five kinds of serotype Coxsackie viruss.
Embodiment
Inventor's process thinks that to the extensive and deep research of the various serotype sequences of Coxsackie virus Coxsackie virus genome VP1 region sequence is particularly suitable for detecting and identifying the Coxsackie virus of multiple serotype.Not only can design the primer of specific amplification Coxsackie virus to this zone, also can design the Coxsackie virus specific probe.Like this, in a PCR reaction,, just can identify Coxsackie virus quickly and easily through the fluorescent signal of detection probes.
Particularly, the inventor has designed five cover primer and probes to five kinds of serotype Coxsackie viruss, and every cover primer and the equal high special of probe detect in corresponding serotype virus.With plasmid control molecule be the detection lower bound analysis of outer standard substance show foundation can detected minimum template amount be 2 copies to five kinds of serotypes virus detection architecture, the Coxsackie virus RT-PCR detection method sensitivity of setting up than forefathers improves at least one one magnitude.The system sensitivity of detection Coxsackie virus of the present invention then improves significantly to and can detect the plasmid control molecule DNA that is low to moderate 2 copies.
Primer
As used herein, term " Coxsackie virus Auele Specific Primer " refer to such primer (to), the amplified production that it amplifies has the nucleotide sequence (for example nucleotide sequence shown in the SEQ ID NO:16-20) in Coxsackie virus genome VP1 district.
On design of primers, in order to satisfy the needs that at present Coxsackie virus detected better, the inventor has selected the VP1 district conduct that degree of variation is littler, sequence homology is higher between the homologous serotype different subtype to detect target sequence.Simultaneously, the inventor compares to the Coxsackie virus cDNA sequence of having announced and analyzes, select with other enterovirus homology low, but conservative relatively one section sequences Design primer and probe between each serotype.
A kind of preferred, primer of being applicable to A9 type Coxsackie virus is to having the sequence shown in SEQ ID NO:1 and 2.
A kind of preferred, primer of being applicable to A16 type Coxsackie virus is to having the sequence shown in SEQ ID NO:4 and 5.
A kind of preferred, primer of being applicable to B2 type Coxsackie virus is to having the sequence shown in SEQ ID NO:7 and 8.
A kind of preferred, primer of being applicable to B3 type Coxsackie virus is to having the sequence shown in SEQ ID NO:10 and 11.
A kind of preferred, primer of being applicable to B5 type Coxsackie virus is to having the sequence shown in SEQ ID NO:13 and 14.
As used herein; Term " Auele Specific Primer "; Refer to that length is the oligonucleotide chain of 22 ± 4Nt, its with five kinds of serotypes (A9 type, A16 type, B2 type, B3 type, B5 type) Coxsackie virus in cDNA sequence behind a kind of serotype viral RNA rt complementary fully perhaps identical.
Probe
As used herein, term " Coxsackie virus specific probe " refers to be incorporated into the amplified production of Coxsackie virus, but debond is in the probe of the amplified production of other species (like pig, ox, sheep or other virus etc.).More preferably, described Coxsackie virus specific probe specificity is incorporated into the nucleotide sequence in Coxsackie virus genome VP1 Coxsackie virus, shown in SEQ ID NO:16-20 district.
A kind of preferred, be applicable to that the probe of A9 type Coxsackie virus has the sequence shown in the SEQ ID NO:3.
A kind of preferred, be applicable to that the probe of A16 type Coxsackie virus has the sequence shown in the SEQ ID NO:6.
A kind of preferred, be applicable to that the probe of B2 type Coxsackie virus has the sequence shown in the SEQ ID NO:9.
A kind of preferred, be applicable to that the probe of B3 type Coxsackie virus has the sequence shown in the SEQ ID NO:12.
A kind of preferred, be applicable to that the probe of B5 type Coxsackie virus has the sequence shown in the SEQ ID NO:15.
Can be used for the not special restriction of probe of the present invention; Can comprise (but being not limited to): Taqman probe and Taqman-MGB probe; Preferred Taqman-MGB probe because the Taqman-MGB probe because have that specificity is good, highly sensitive, good stability, optimization step is simple, the result is accurate, the resolving power advantages of higher.Simultaneously, the length of Taqman-MGB probe design is shorter than general T aqman probe, can be less than 20bp.In addition, the Tm value difference that the Taqman-MGB probe can improve between pairing and non-matching template is different, and when between non-matching template and pairing template the difference of a base being arranged, this probe does not promptly combine with it and can not carry out pcr amplification, thereby improves the specificity that detects greatly.
As used herein, term " specificity T aqman-MGB probe " refers to that length is the oligonucleotide chain of 18 ± 4Nt, and its 5 ' end mark FAM fluorescence excitation group, and 3 ' end mark the MGB fluorophor; Its with five kinds of serotype Coxsackie virus RNA rts after the cDNA sequence fully complementary or identical; It combines to form a cover real-time fluorescence PCR reaction system with Auele Specific Primer.
Should be understood that the use of specificity T aqman-MGB probe and Auele Specific Primer coupling.For example, when A9 type Coxsackie virus is detected, select for use SEQ ID NO:1 and SEQ ID NO:2 as primer, SEQ ID NO:3 implements pcr amplification as probe.
The Taqman-MGB probe can be told the difference of a base of template and probe land, and detection specificity is high, and the MGB probe length is very favourable for seeking suitable surveyed area between the higher virus strain of sudden change degree 13-19 base.Use MGB probe of the present invention can realize the sensitivity of higher detection Coxsackie virus.
MGB probe of the present invention has following advantage:
(1) length that shortens probe is to 14-19bp, and the designs specificity detection probes has very great help between the same serotype different virus strain big to degree of variation, that mutation rate is higher;
(2) the Taqman-MGB probe is different through the Tm value difference that improves between pairing and non-matching template, and the distinguishable difference that goes out a base of template and probe land is particularly suitable for GC content sequence on the low side, and detection specificity is significantly improved;
(3) MGB is not luminous fluorophore FAM and reports its position in space group closer butt off with TAMRA group, compared with the lower end, a higher signal to noise ratio, higher stability , more accurate results, the advantages of higher resolution.
(4) MGB probe optimum experimental step is simple, good stability, and repeatability is high.
Object of reference
The present invention also provides and has been used for the marker (plasmid control molecule) that Coxsackie virus detects.Described marker is Coxsackie virus cDNA, or contains the Coxsackie virus amplification segmental DNA of purpose (plasmid control molecule).DNA that preferred marker is Coxsackie virus genome VP1 district or the segmental DNA of its amplification purpose.
For example, object of reference can be to contain five kinds of cDNA behind the serotype Coxsackie virus RNA rt respectively, perhaps contains the plasmid of said cDNA.These markers can be used this area ordinary method preparation.
Detection method
The present invention also provides the method that is used for detecting simultaneously five kinds of serotype Coxsackie viruss.For example, the primer and the probe that five pairs of Auele Specific Primers shown in the SEQ ID NO:1-15 and corresponding five specificity T aqman-MGB probes are used separately as pcr amplification reaction.
Real-time fluorescence PCR is airtight amplification detection method, after the amplification system application of sample finishes, can increase and detect, and does not analyze after need not increasing again; Not only reduce the possibility of environmental pollution, also owing to there is primer pair to hybridize jointly, higher than regular-PCR specificity with probe.
The TaqMan polymerase chain reaction technique utilizes Taq enzyme 5 ' → 3 ' 5 prime excision enzyme activity, in the process of extending, realizes the cut-out to hybridization probe, eliminates the cancellation effect of probe 3 ' end fluorophor to 5 ' end mark report fluorophor signal.The quantity of the power of fluorescent signal and PCR product is proportional, and detection system can be inferred the quantity of PCR product through detecting fluorescence.
Preferred amplification region is the nucleotide sequence in Coxsackie virus genome VP1 Coxsackie virus, shown in SEQ ID NO:16-20 district in the inventive method, and this zone is that Coxsackie virus is distinctive, can be used for detecting Coxsackie virus.
According to instruction of the present invention, those skilled in the art can design and synthetic primer and Coxsackie virus specific probe, and is used for fluorescent real time PCR qualitative and quantitative detection technology.
In the present invention; Except the primer that detects the target sequence be directed against and corresponding adopted (to) with probe etc. with prior art is different; Condition such as from steps such as the total extracting RNA of sample, pcr amplification and fluoroscopic examinations can be identical with prior art, and those skilled in the art can carry out above-mentioned various step with the condition of routine fully.Certainly, also can adopt optimum condition given among the application embodiment.
For example, can be used for viral enrichment of the present invention, RNA extractive technique and method and have no particular limits, can be that this area is used more, good stability, various extractive techniques that safety is high.A kind of preferred method is PEG8000 enrichment and Trizol RNA extraction method.
In a preferred embodiment of the invention, electrophoresis result shows that the Auele Specific Primer of amplification Coxsackie virus is to increasing to the nucleic acid of Coxsackie virus.The real-time fluorescence detected result shows, only containing the sample performance positive of Coxsackie virus, produces a desired effect.This shows that the Coxsackie virus specific probe that goes out through choose reasonable is very effective and special.
When using the quantitative PCR appearance to carry out the real-time fluorescence detection,, can detect at 530nm or 640nm equiwavelength according to the difference of fluorescent marker (FAM, TET).
In a preference, the present invention adopts the real-time fluorescence PCR detection technique through five pairs of primers and five probes of design, detects and identify all five kinds of serotype Coxsackie viruss.Can detect five kinds of serotypes (A9 type, A16 type, B2 type, B3 type, B5 type) Coxsackie virus respectively through using paired primer and probe.Whether can send tangible detectable signal and blank does not have under the detectable signal situation at the marker of reaction design, whether implement the real-time fluorescence PCR amplification per sample has tangible detectable signal judgement sample polluted by Coxsackie virus.
In another preference, a kind of Coxsackie virus real-time fluorescence PCR detection method can specifically comprise the steps:
1.. the extraction of Coxsackie virus RNA;
2.. Coxsackie virus RNA rt is cDNA;
3.. with cDNA is template, carries out pcr amplification respectively with (a) said Auele Specific Primer and (b) said Taqman-MGB probe, and measures the detectable signal that produces.Wherein, described Auele Specific Primer is to one section sequence in five kinds of serotype genomes of Coxsackie virus VP1 district that increases specifically, and described Taqman-MGB probe combines with corresponding primer amplified product specificity;
4.. according to the detectable signal assay determination data of collecting, thereby confirm and detect whether there are above-mentioned five kinds of serotype Coxsackie viruss in environmental sample and the human excrement sample.
In another preference, step 3. in, measuring detectable signal is in the real-time fluorescence PCR amplified reaction, the fluorescent signal that the reporter group FAM of probe is produced; 3. also comprise with marker and distilled water as template the pcr amplification that carries out respectively with (a) said Auele Specific Primer and (b) said Taqman-MGB probe in step; And step comprise in 4. to the amplification assay of marker and distilled water with collect detectable signal, the line data analysis of going forward side by side.Wherein, described marker is Coxsackie virus cDNA or contains the segmental plasmid control molecule DNA of Coxsackie virus amplification purpose.
Major advantage of the present invention is:
(1) can detect and identify the Coxsackie virus in environmental sample and the human excrement sample easy, apace.
(2) based on the primer in Coxsackie virus genome VP1 district to the nucleotide sequence of specific amplification Coxsackie virus effectively, the Coxsackie virus specific probe then can be distinguished Coxsackie virus and other viruses more specifically.
(3) the detected result good linearity can be used for quantitative analysis.
(4) detection method sensitivity improves at least one one magnitude.The detection lower limit of five kinds of serotype Coxsackie virus detection architecture all can be low to moderate 2 copy plasmid control molecule DNA.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to people such as normal condition such as Sambrook; Condition described in the molecular cloning laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber are weight percent and parts by weight.
1 materials and methods
1.1 material and reagent
The TIANamp viral RNA extracts test kit: day root bio tech ltd, Cat.No.SD101; Prime Script
TMReverse transcription test kit: precious biotechnology ltd, Cat.No.DRR037A; Premix Ex Taq
TMReal-time fluorescence amplification kit: precious biotechnology ltd, Cat.No.DRR039A.
1.2 primer and probe
In the GenBank DB, search CA 9, A16, B2, B3 and B5 type cDNA sequence respectively, and compare and analyze, seek in each serotype conservative relatively, but with other serotype Coxsackie virus and the lower zone of enterovirus homology.Select Coxsackie virus genome coat protein VP1 coding region to design primer and probe CoxA9-F/R/P, CoxA16-F/R/P, QCoxB2-F/R/P, QCoxB3-F/R/P and QCoxB5-F/R/P respectively, be suitable for the detection of CA 9, A16, B2, B3 and B5 type respectively.Primer and probe sequence information are seen table 1.After carrying out the Blast comparison, designed primer and probe only mate with the Coxsackie virus correlated series.
The common RT-PCR of table 1 Coxsackie virus detects primer
Wherein, S:C/G; M:A/C; W:A/T; Y:T/C; V:A/C/G; R:A/G; D:A/G/T; K:G/T.
1.3 viral RNA extracts in viral inactivation vaccine, the faecal samples
1.4 reverse transcription reaction
Adopt Prime Script
TM(precious biotechnology (Dalian) ltd Cat.No.:DRR037A) carries out reverse transcription reaction to the reverse transcription test kit.Reaction system is 10 μ L, comprises 1 * Buffer, 0.5 μ L RT Enzyme Mix, 0.5 μ L Oligo (dT) (0.125 μ M), 5 μ L RNA solution.Reaction conditions is: 37 ℃, and 15min; 85 ℃, 5s.
1.5PCR reaction
1.5.1 common PCR reaction
((Shanghai) bio tech ltd spins in Japan, Cat.No.:BTQ-201) to use the regular-PCR test kit.The PCR reaction system is 25 μ L.Comprising 1 * PCR damping fluid, 1.25U BlendTaq-Plus archaeal dna polymerase, 0.4 μ M upstream primer, 0.4 μ M downstream primer, 0.2mM dNTPs, 2 μ L cDNA solution.Primer EV1/2
[10]Reaction conditions is: 94 ℃, and 2min; 94 ℃, 30s, 60 ℃, 1min, 72 ℃, 1min; 45 circulations.Primer GII-SKF/R
[11]Reaction conditions is: 94 ℃, and 2min; 94 ℃, 30s, 50 ℃, 30s, 72 ℃, 1min; 45 circulations.
Get PCR product 15 μ L respectively, add 1.5 μ L, 10 * sample-loading buffer point sample, and (precious biotechnology (Dalian) ltd, D501A) point sample is to judge the clip size of PCR product to add dna molecular amount mark DL 2000.Electrophoresis detection result is with gel analysis imaging system record.
1.5.2 real-time fluorescence PCR reaction
Use Premix Ex Taq
TM(precious biotechnology (Dalian) ltd Cat.No.:DRR039A) carries out the real-time fluorescence PCR reaction to test kit, and reaction system is seen table 2.Reaction conditions is: 94 ℃, and 10s; 94 ℃, 5s, 60 ℃, 31s, 45 circulations.Multiple 3 times of institute's counterpoise that responds.As positive control, as negative control, replace template as blank with Coxsackie virus RNA or its cDNA with water with the RNA that do not contain Coxsackie virus or cDNA.
The reaction system that table 2 Coxsackie virus real-time fluorescence RT-PCR detects
Annotate
*: when A16 type Coxsackie virus was detected, primer and probe were respectively QCoxA16-F/R/P; When B2 type Coxsackie virus was detected, primer and probe were respectively QCoxB2-F/R/P; When B3 type Coxsackie virus was detected, primer and probe were respectively QCoxB3-F/R/P; When B5 type Coxsackie virus was detected, primer and probe were respectively QCoxB5-F/R/P.
1.6 plasmid control molecule makes up
Adopting primer QCoxA9-F/R, QCoxA16-F/R, QCoxB2-F/R, QCoxB3-F/R and QCoxB5-F/R respectively is that template is carried out regular-PCR amplification (as: primer CoxA9-F/R is a template with CA 9 type cDNA) with target serotype virus cDNA in the table 1, and the purpose clip size is respectively 91bp, 174bp, 76bp, 80bp and 145bp (SEQ ID NO:16-20 and Fig. 2).Reclaim the above fragment of purifying, be cloned into (precious biotechnology (Dalian) ltd) in the pMD-18T carrier, cut and the definite positive colony of the evaluation of checking order through enzyme.The plasmid control molecule pMD-A9, pMD-A16, pMD18-B2, pMD18-B3 and the pMD18-B5 that are contained CA 9, A16, B2, B3 and B5 type VP1 district specific fragment respectively.In order to set up typical curve, five kinds of plasmid standard molecules that make up are diluted to 10 respectively
6, 10
5, 10
4, 10
3, 10
2, 10 copy/μ L, and further be diluted to 5,1 copy/μ L and be used to detect lower bound analysis.
The position relation of primer and amplified production is as shown in table 3 below:
Table 3
2. result
2.1 the Coxsackie virus RT-PCR detection architecture specificity of setting up test
In order to test the Coxsackie virus real-time fluorescence RT-PCR detection architecture specificity of foundation; With A9, A16, B2, B3 and B5 type Coxsackie virus, I, II and III type poliovirus and GII type norovirus cDNA is template; Adopt primer and probe QCoxA9-F/R/P, QCoxA16-F/R/P, QCoxB2-F/R/P, QCoxB3-F/R/P, QCoxB5-F/R/P respectively, to carry out the real-time fluorescence PCR amplification with top plate.The result shows that CA 9 type detection architecture amplification curve occurs when only being template with CA 9 type cDNA, all do not see amplified signal when being template with other serotype Coxsackie virus, poliovirus and norovirus cDNA; Likewise, other four kinds of serotype Coxsackie virus RT-PCR detection architecture only the fluorescence width of cloth occurs and increase in the reaction of amplification target serotype virus cDNA, all do not see amplified signal (table 4) when being template with other viral cDNA.
For avoiding false negative, detect primer EV1/2 with enterovirus
[10]Coxsackie virus and poliomyelitis virus cdna sample are detected the purpose fragment that all can increase and obtain 435bp.GII type norovirus cDNA sample is used primer GII-SKF/R
[11]Increase, obtain the purpose fragment (data do not appear at this) of 344bp, show that above-mentioned viral RNA all successfully separates and is inverted record is cDNA, can be used for pcr amplification.Therefore, primer and probe QCoxA9-F/R/P high special detect in A9 type Coxsackie virus real-time fluorescence RT-PCR; Primer and probe QCoxA16-F/R/P, QCoxB2-F/R/P, QCoxB3-F/R/P, QCoxB5-F/R/P are specific to the detection of target serotype Coxsackie virus similarly respectively.
The specificity test of table 4 real-time fluorescence RT-PCR detection architecture
Annotate: "+" expression detected result is positive; "-" expression detected result is negative.
2.2 Coxsackie virus real-time fluorescence RT-PCR detection architecture standard curve making
Respectively plasmid control molecule pMD18-A9, pMD18-A16, pMD18-B2, pMD18-B3 and pMD18-B5 are diluted to 10
6, 10
5, 10
4, 10
3, 10
2, 10 copy/μ L; Carry out the real-time fluorescence PCR reaction; Each gradient template triplicate reaction prepares A9, A16, B2, B3, B5 type Coxsackie virus RT-PCR detection architecture typical curve (Fig. 1) respectively, and average Ct value of each gradient and corresponding standard deviation thereof are seen table 5.
Can find out the good (R of typical curve linear relationship of five kinds of serotype virus detection architecture by Fig. 1, table 5
2＞0.999).Five kinds of serotype Coxsackie virus regression equation slopes are respectively-3.33 ,-3.17 ,-3.34 ,-3.33 and-3.14, according to PCR reaction efficiency formula (E=10
-1/slope-1, slope is a slope) calculate, get reaction efficiency and be respectively: 99.25%, 99.66%, 107.97%, 99.66% and 106.54%; The Ct value of each gradient template at interval evenly; Standard deviation (SD) all is lower than 0.218, and is repeatable good, meets the kinetics requirement of PCR reaction.Therefore, above result shows that the detection architecture that the present invention sets up is applicable to the sample quantitative analysis.
The repeatable test of five kinds of serotype plasmid control molecules of table 5 Coxsackie virus detection architecture
2.3 the Coxsackie virus real-time fluorescence RT-PCR detection architecture sensitivity analysis of setting up
In order to analyze the detection architecture sensitivity of foundation; With 5,1 copy/μ L plasmid control molecule pMD18-A9 is template; Each concentration respectively repeats ten times, and ten detections the fluorescence width of cloth all occurs and increase, and the above plasmid control molecule DNA of 1 copy/μ L concentration template all detects tangible amplification as a result.When being template, obtain analog result, all can detect the tangible fluorescence width of cloth when promptly the above concentration of 1 copy/μ L is template and increase with 10 times of gradient dilution liquid of plasmid control molecule pMD-B3, pMD-B5, pMD-A9 and pMD-A16.Therefore, the detection lower limit of the real-time fluorescence RT-PCR detection architecture that is suitable for five kinds of serotype Coxsackie viruss detections of foundation all can reach 2 copies (the template amount is 2 μ L).
3 discuss
For Coxsackie virus is implemented effective monitoring, set up that the Coxsackie virus detection method is most important fast and effectively.Nearly 30 kinds of Coxsackie virus serotypes, and the genome mutation degree is bigger between type and the type, and it is very difficult therefore to design the general detection primer that is suitable for all serotypes of Coxsackie virus, and relevant both at home and abroad at present research is reported also less.According to characteristics conservative relatively in each serotype of Coxsackie virus, the present invention is directed to CA 9, A16, B2, B3 and B5 type and designed a cover primer and a probe respectively.For high specific and the sensitivity that guarantees to detect, the present invention has selected the Taqman-MGB probe to be used for detecting.Through test, five cover primers and probe QCoxA9-F/R/P, QCoxA16-F/R/P, QCoxB2-F/R/P, QCoxB3-F/R/P and QCoxB5-F/R/P all are specific to the detection of corresponding serotype Coxsackie virus.
Based on the CA that has made up 9, A16, B2, B3 and B5 type plasmid control molecule; The present invention carries out sensitivity analysis to five kinds of serotype virus detection architecture; Obtain the sensitivity of five kinds of serotype virus detection architecture and be 2 copy DNAs, the good (R of the typical curve linear relationship of making
2＞0.999).(2003) such as Kageyama T.
[12]The norovirus real-time fluorescence RT-PCR detection architecture sensitivity of setting up is 10 copy DNAs, typical curve coefficient of determination R
2=0.995/0.998; Show that from above interpretation of result detection architecture sensitivity and typical curve result that the present invention sets up are very excellent.
Therefore, what the present invention set up has the good and highly sensitive characteristics of specificity to CA 9, A16, B2, B3 and B5 type real-time fluorescent RT-PCR method for detecting respectively, can satisfy the requirement of routine check quarantine.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Reference
[1] Chen Shuxia thanks to Longshan, Mei Shangwen, etc. CB RNA and neutralizing antibody detect the meaning [J] in the viral myocarditis diagnosis. Chinese laboratory medicine magazine 2003,26 (3): 166-168.
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Hong Ling,
Lei Xianying, etc. the epidemiology survey [J] that breaks out of CB 5 types together.
China's epidemiology magazine, 2008,29 (5): 112-113,116.
[3] Shi Xian, Liu Kehong. the diagnosis and treatment analysis [J] of Coxsackie virus eruption and prevalence together. the practical medical science in Zhejiang, 2004,02:126-127.
[4] Gaogang District board of health website: www.ggwsj.gov.cn/UploadFile/200805/12/1036166845.doc
[5]Tana?E.L.，Chowa?V.T.K.，Quakc?S.H..Development?of?multiplex?real-time?hybridization?probe?reverse?transcriptase?polymerase?chain?reaction?for?specific?detection?and?differentiation?of?Enterovirus?71?and?Coxsackievirus?A16[J].Diagnostic?Microbiology?and?Infectious?Disease，2008，61：294-301.
[6] Justin W.A.B.; Patrick S.O.; Deng people Serotype-specific detection of Coxsackievirus A16 in clinical specimens by reverse transcription-nested PCR [J] .J Clin Micro; 2001,39 (10), 3690-3692.
[7] Shi Wen, Lu Yiyu, Yan Juying, etc. Zhejiang Province's coxsackie B 3 virus VP 1 district genetic analysiss [J] in 2006. Chinese sanitary inspection magazine, 2008,18 (4): 599-601.
[8] Swanink C.M., Veenstral L., Poort Y.A.; Deng people .Coxsackievirus B1-based antibody-capture enzyme-linked immunosorbent assay for detection of immunoglobulin G (IgG); IgM, and IgA with broad specificity for enteroviruses [J] .J Clin Microbiol, 1993; 31 (12), 3240-3246.
[9]Kutyavin?L.V.，Afonina?I.A.，Mills?A..3’Minor?groove?Binder-DNA?probes?increase?sequence?specificity?at?PCR?extension?temperatures[J].Nucleic?Acids?Research，2000，Vol.28(2)：655-661.
[10] Andreoletti L.; Hober D.; Belaich S.; Deng people .Rapid detection of enterovirus in clinical specimens using PCR and microwell capture hybridization assay [J] .J Virol Methods, 1996,62 (1): 1-10.
[11] Kojima S., Kageyama T., Fukushi S. waits people .Genogroup-specific PCR primers for detection of Norwalk-like virus [J] .J Virol Methods, 2002,100 (1-2): 107-114.
[12] Kageyama T.; Koj ima S.; Shinohara M.; Deng people .Broadly reactive and highly sensitive assay for Norwalk-Like viruses based on real-time quantitative reverse transcription-PCR [J] .J Clin Micro, 2003,41 (4): 1548-1557.
Claims (10)
1. polymerase chain reaction method; It is characterized in that; It comprises step: in the polymeric enzyme reaction system, carry out the polymerase chain reaction; The Auele Specific Primer that contains the Coxsackie virus that increases in the described reaction system to the Coxsackie virus specific probe, and the amplified production that goes out of said primer amplification has the nucleotide sequence in Coxsackie virus genome VP1 district.
2. the method for claim 1 is characterized in that, the Auele Specific Primer of described amplification Coxsackie virus is right to comprising the 1-5 kind primer that is selected from down group:
A9 type Coxsackie virus: SEQ ID NO:1 and SEQ ID NO:2;
A16 type Coxsackie virus: SEQ ID NO:4 and SEQ ID NO:5;
B2 type Coxsackie virus: SEQ ID NO:7 and SEQ ID NO:8;
B3 type Coxsackie virus: SEQ ID NO:10 and SEQ ID NO:11;
B5 type Coxsackie virus: SEQ ID NO:13 and SEQ ID NO:14.
3. the method for claim 1 is characterized in that, described Coxsackie virus specific probe is Taqman-MGB probe or Taqman probe.
4. like arbitrary described method among the claim 1-3, it is characterized in that described Coxsackie virus specific probe has the nucleotide sequence of the group of being selected from down:
A9 type Coxsackie virus: SEQ ID NO:3;
A16 type Coxsackie virus: SEQ ID NO:6;
B2 type Coxsackie virus: SEQ ID NO:9;
B3 type Coxsackie virus: SEQ ID NO:12;
B5 type Coxsackie virus: SEQ ID NO:15.
5. like arbitrary described method among the claim 1-3, it is characterized in that said method also comprises step: in the process of polymerase chain reaction or afterwards, detect the fluorescent signal that the Coxsackie virus specific probe sends.
6. a detection kit is characterized in that, it contains following reagent:
(a) Auele Specific Primer of amplification Coxsackie virus is right, and the amplified production that goes out of said primer amplification has the nucleotide sequence in Coxsackie virus genome VP1 district; With
(b) specific probe of detection Coxsackie virus.
7. test kit as claimed in claim 6 is characterized in that, the Auele Specific Primer of described amplification Coxsackie virus is right to comprising the 1-5 kind primer that is selected from down group:
A9 type Coxsackie virus: SEQ ID NO:1 and SEQ ID NO:2;
A16 type Coxsackie virus: SEQ ID NO:4 and SEQ ID NO:5;
B2 type Coxsackie virus: SEQ ID NO:7 and SEQ ID NO:8;
B3 type Coxsackie virus: SEQ ID NO:10 and SEQ ID NO:11;
B5 type Coxsackie virus: SEQ ID NO:13 and SEQ ID NO:14.
8. test kit as claimed in claim 6 is characterized in that, described Coxsackie virus specific probe is Taqman-MGB probe or Taqman probe.
9. like arbitrary described test kit among the claim 6-8, it is characterized in that described Coxsackie virus specific probe has the nucleotide sequence of the group of being selected from down:
A9 type Coxsackie virus: SEQ ID NO:3;
A16 type Coxsackie virus: SEQ ID NO:6;
B2 type Coxsackie virus: SEQ ID NO:9;
B3 type Coxsackie virus: SEQ ID NO:12;
B5 type Coxsackie virus: SEQ ID NO:15.
10. the purposes of test kit as claimed in claim 6 is characterized in that, is used for detecting whether there is Coxsackie virus at environmental sample or human excrement sample.
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| CN102816870B (en) * | 2012-08-31 | 2014-04-09 | 何雅青 | Primer and kit for detecting coxsackievirus A6 type RT-LAMP (Reverse Transcription Loop-mediated Isothermal Amplification) nucleic acid |
| CN106282412A (en) * | 2016-10-08 | 2017-01-04 | 陕西脉元生物科技有限公司 | A kind of based on loop-mediated isothermal amplification technique three kinds of enterovirus test kits of detection and detection method |
| CN108531661A (en) * | 2018-06-08 | 2018-09-14 | 东莞市第八人民医院 | A kind of fluorescence quantification PCR primer, probe, kit and the method for detection CVB3 |
| CN109897919A (en) * | 2019-04-23 | 2019-06-18 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | Coxsack A6 type, the method and kit of A10 type are diagnosed simultaneously |
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| CN110592287A (en) * | 2019-10-23 | 2019-12-20 | 复旦大学附属中山医院 | Coxsackie group B virus real-time fluorescent quantitative PCR standard substance |
| CN111748650A (en) * | 2020-06-23 | 2020-10-09 | 深圳市艾伟迪生物科技有限公司 | Nucleic acid composition for simultaneously detecting four B-group coxsackie virus subtypes and kit and detection method thereof |
| CN112593011A (en) * | 2020-12-25 | 2021-04-02 | 中山大学 | Primer and probe for detecting coxsackie virus B group |
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