CN101880732B - Simian parainfluenza virus 5 RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method and kit - Google Patents
Simian parainfluenza virus 5 RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method and kit Download PDFInfo
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Abstract
The invention discloses an SV5 virus RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method and a kit. A primer is designed by taking M gene segment nucleic acid as a detected object and used for quantificationally detecting the copy number of nucleic acid of an SV5 virus in a sample. The detection method comprises the following steps of: (1) extraction of a sample RNA; (2) reverse transcription of the RNA; (3) RCR detection: carrying out PCR amplification under the guide of the primer by taking the reverse transcription product of the virus RNA obtained in the step (2) as a template, and carrying out agarose gel electrophoresis detection after the reaction is ended, if the target segment of 317bp can be amplified, showing that the detection result represents SV5 positive, otherwise, showing that detection result represents SV5 negative. The invention has great actual significance in the detection field of SV5 in animal-based biological products or relevant raw materials and animals and has wide application potential.
Description
Technical field
The present invention relates to the detection method of virus in the biological technical field, particularly relate to the RT-PCR detection method and the test kit of monkey parainfluenza virus type 5 (SV5).
Background technology
Monkey parainfluenza virus type 5 (Simian parainfluenza virus 5; SV5) be to separate from the MK cells culture by people such as Hull first in 1956 to obtain; In animal bodies such as people, dog, also be separated to this virus subsequently in succession; SV5 belongs to Paramyxoviridae in classification, paramyxovirus genus, and other member of generic comprises human parainfluenza virus 1-4 type, mumps virus, NDV, Sendai virus etc.
SV5 often exists with the mode of inapparent infection in the monkey crowd; Many no clinical symptom; The SV5 antiviral antibody that in cavy, suslik, dog, cow, goat and sheep serum, also contains height in addition; Antibody positive rate reaches 20% in cavy, and antibody positive rate reaches 43% in the suslik crowd, shows the infection that has SV5 virus or correlated virus.SV5 extensively is present among the monkey crowd in Asia and Africa, and is the most common with rhesus monkey especially.In the monkey crowd of countries such as Malaysia and Cambodia, the separation rate of SV5 is up to 30-33%, and antibody positive rate is up to 70-80%.At present China does not see that as yet monkey infects the report of SV5, and SV5 also can infected person, but does not see that the clinical symptom report is arranged.A situation arises in other fauna it be unclear that for SV5.
SV5 is nucleic acid based to be single-stranded RNA, and virus particle is rounded, and diameter 150-250nm includes the symmetric nucleoprotein of type in the shape of a spiral, and diameter 15-18nm is peripheral with the lipoprotein cyst membrane, contains fine dashing forward on the cyst membrane, is the hemagglutinin and the neuraminidase part of virus.SV5 can breed on the cells such as Vero at former generation of multiple animals such as monkey, people, baboon, ox, dog, hamster and cavy kidney cell and BHK-21, wherein with former generation MK cells susceptible the most, and can produce the acidophilia intracytoplasmic inclusion.Mostly the cytopathy that SV5 causes is to form the multinuclear nuclear inclusion cell that a plurality of nuclears flock together, and cavity ringwise, and some cell is fusiformis.
SV5 and human parainfluenza virus 1-4 type all have antigenic cross-reaction, with SV-41, mumps virus (mumps), Measles virus (measles) cross reaction are arranged also.Virus can be present in the histoorgan of monkey (especially kidney), therefore usually causes the pollution of monkey source cell culture.With SV5 via intranasal application artificial challenge rhesus monkey, can from throat swab, find virus behind the 3d, also separable to virus from lung and tracheae behind the 7d, but infect the no any symptom performance in back.Experiment condition infects cavy down can not cause tangible disease, but can stimulate body to produce antibody.Infect very susceptible of hamster in the intranasal, can produce symptoms such as encephalitis.
SV5 is the RNA viruses of the non-segmented negative of strand, negative justice; The key character that it is different from other paramyxovirus family viral is exactly that it has the minimum viral genome of a cover; Total length 15246nt is by 3 ' end homing sequence (55nt) and 5 ' end sequence (31nt) and seven sections successional genomic constitutions.They are respectively: L gene (6804nt, coding polymerase protein large polymerase protein), HN gene (1789nt, coding blood clotting-neuraminidase hemagglutinin-neuraminidase); SH gene (292nt, little hydrophobic proteins smallhydrophobic protein encodes), F gene (1873nt; Encoding fusion protein fusion protein), M gene (1371nt, coding membrane matrix albumen; Matrix protein), P gene (1298nt, coding phosphorylated protein; Phosphoprotein), NP gene (1725nt, coding virus nucleocapsid albumen neucleocapsid protein).Wherein P gene 3 ' end has a bit of gene codified V protein again, and this a bit of gene is called V gene (669nt) again.164 amino-acid residues of N-terminal are identical with P albumen (392 residues) for V protein (222 residues), but C-terminal is different.SV5 on the genome structure and Sendai virus (SeV), Stomatovirus important differences of virus such as (VSV) be exactly: the joint portion has highly variable between each gene of SV5 genome, and viruses such as SeV, VSV are then quite conservative.Research recently shows that the joint portion is being differentiated aspect regulation and control polysaccharase function and the decay between each gene of SV5.
NP albumen is the chief component of nucleocapsid protein, divides two main region: the one, and amino acid region, it directly combines with viral RNA; Another is carboxyl terminal zone, is exposed to the nucleocapsid surface after the assembling, has protease-sensitive binding site.P albumen, V protein, L albumen belong to the complementary nucleus capsid protein; Be and rna replicon and the relevant main complementary albumen of enzymatic reaction process of transcribing; The collaborative each other competence exertion effect of these accessory proteins, P albumen and V protein can interact and form virus replication and the polymerase mixture of transcribing.V protein can make NP albumen keep solubility before the effect of dressing shell, and to prevent the NP proteolyze, V protein can also stop NP albumen and P albumen to form different aggressiveness before viral RNA is synthetic.Polysaccharase L albumen can be transcribed at a fragment gene exactly one of effect of virus replication and closed to an end and next fragment gene is transcribed and stopped transcribing of this gene section when not beginning as yet, and adds the polyA tail and its 5 ' end is modified at the corresponding mRNA3 ' of this gene end.And the proteic synthetic transcriptase that receives of L is through the precision regulation and control of efficient at interval of M-F gene; Because in case transcriptase improves through M-F gene interval efficient then can be with the overexpression that causes downstream L gene; This will cause the viral growth defective, and this point is very important for the optimization growth of virus particle.
HN albumen, F albumen, M albumen and SH albumen all belong to the membrane glycoprotein of virus, and adhering to of protein mediated virus of HN and cell has hemoglutinin and neuraminic acid enzymic activity; F albumen is fusion rotein, and it can promote virus envelope and host cell surface lipoprotein membrane to merge; M albumen is present in the internal layer of virus envelope, is a kind of less relatively non-glycosylated protein, and it is participated in cyst membrane and forms in the virus particle assembling process, and the identification between mediation nucleocapsid and the cyst membrane is to keep the integrity of virus particle structure; SH albumen (5.012KD, 44 amino acid) is a little hydrophobic proteins, and concrete function it be unclear that, and possibly affinity arranged to bilayer lipid membrane.
Because SV5 virus is inapparent infection more in the monkey crowd; Infected monkey but do not fall ill; Can the biological products of monkey source cell production be polluted; Though the present domestic report that has monkey to infect SV5 of not seeing is as yet set up complete SV5 cause of disease and the antibody test means of a cover and is very important in the quality control of laboratory animal and biological products.For the mainly still separation and the evaluation of virus of specificity diagnostic method of SV5 virus infection, specifically can be divided into following two big types of detection methods at present:
(1) Detection of antigen tissue culture observation of cell pathology, hemagglutination test, hemabsorption, the virion in the electron microscopic examination cell culture.
(2) the isolating viral available standards antiserum(antisera) of antibody test does that blood clotting suppresses, methods such as hemocyte absorption inhibition, serum neutralization test, immunofluorescent test and ELISA identify.Conventional sense SV5 antibody generally with the comparatively special sensitivity of ELISA method, can select for use another kind of method to verify when positive findings occurring, perhaps verifies with the ELISA-blocking experiment.
But still there is the limitation of aspects such as specificity and sensitivity in these methods.
Summary of the invention
To above problem, the contriver proposes a kind of RT-PCR method through the detection of nucleic acids of SV5 virus is studied; And expectation is easy and simple to handle with it; Specificity and sensitivity are higher, are easy to advantages such as popularization, in the early diagnosis that it is applied to virus solution being provided.
Therefore, purpose of the present invention provides and is used for SV5 virus is carried out the primer that RT-PCR detects.
Primer provided by the present invention is that the M gene section nucleic acid with SV5 designs for detected object, in order to the nucleic acid copy number of SV5 virus in the detection by quantitative sample.
Specifically, the upstream primer of said primer has the nucleotide sequence of SEQ ID NO:1 in the sequence table, and downstream primer has the nucleotide sequence of SEQ ID NO:2 in the sequence table.
Second purpose of the present invention provides specific specificity and the higher viral RT-PCR detection method of SV5 of sensitivity.
Detection method provided by the present invention is the RT-PCR detection method of carrying out with primer of the present invention.
Specifically, said RT-PCR detection method can may further comprise the steps:
1) extraction of sample rna:
2) rt of RNA:
The rt product of the viral RNA that 3) PCR detection: with step 2) obtains is a template; Under the right guiding of above-mentioned primer, carry out pcr amplification; Reaction finishes laggard row agarose gel electrophoresis and detects, if can amplify the purpose fragment of 317bp, then detected result is the SV5 positive; Otherwise be the SV5 feminine gender, so that SV5 is carried out qualitative detection.
In above-mentioned RT-PCR detection method, the sample in the said step 1) can be animal derived biological products, blood, in vitro tissue or cell culture etc.
Step 2) being in suitable reaction system, through the catalysis of reversed transcriptive enzyme, is the process of template synthetic DNA chain (cDNA) with RNA; Said 25ul reverse transcription reaction system is: the dH that removes Rnase
2O 7 μ l, 5 * buffer (Promega AMV Reverse Transcriptasse, catalog#M5108) 5 μ l; DNTP (concentration 2.5mM) 4 μ l; Random primer (concentration 500 μ g/mL) 0.5 μ l, RNA (concentration 0.25 μ g/ μ l) 8 μ l, AMV (concentration 10u/ μ l) 0.5 μ l; Said reverse transcription reaction condition is: 37 ℃ of 90min, 72 ℃ of 15min, 4 ℃ of 5min.
Step 3) is that the rt product with viral RNA is a template; In suitable reaction system; Combine with special primer of the present invention and viral genome cDNA corresponding position are complementary; Through sex change (the genome two strands is separated into strand), annealing (primer combines with template), the cycle repeats in 3 stages of extension (from the synthetic new chain of primer 3 ' end) of DNA, the purpose fragment is exaggerated, and carries out the qualitative detection of SV5 virus again through agarose gel electrophoresis.
Said 20ul PCR reaction system can comprise: 10 * buffer (TaKaRa Taq
TMHot Start VersionDR007A) 2 μ l, dNTP (concentration 2.5mM) 1.6 μ l, dH
2O 12.9 μ l, upstream primer (concentration 10 μ M) 1 μ l, downstream primer (concentration 10 μ M) 1 μ l, cDNA (concentration 50ng/ μ l) 1 μ l, Hs Taq enzyme (concentration 5u/ μ l) 0.5 μ l.
Said PCR reaction conditions can be: 94 ℃ of 5min of elder generation; 94 ℃ of 30s then, 58 ℃ of 30s, 72 ℃ of 1min, totally 35 circulations; Last 72 ℃ of 5min.
The 3rd purpose of the present invention provides a kind of test kit that is used for SV5 virus is carried out the RT-PCR detection.
Test kit provided by the present invention comprises the above-mentioned primer that is used for SV5 virus is carried out the RT-PCR detection.
The invention provides a kind of RT-PCR detection method of SV5 virus.This method is to be sensitivity and specificity quite good detecting method all of detected object with M gene nucleic acid in the SV5 genome.The present invention has the following advantages:
1. set up the RT-PCR detection method of SV5 first, with this detection method can the pair cell culture, blood, animal tissues and animal derived biological products carry out high-sensitive detection.
2. this detection method is compared with ELISA with other conventional sense method of SV5 such as separation and Culture evaluation, the agarose diffusion experiment of virus, has quite high sensitivity.
3. other conventional sense method of this detection method and SV5 is compared with competitive ELISA like separation and Culture evaluation, the agarose diffusion experiment of virus, has quite high specificity.
4. this detection method and other conventional sense method, as the separation and Culture of virus identify, the agarose diffusion experiment compares with competitive ELISA, has the advantages that schedule of operation is simple and easy to usefulness, can realize sequencing, suit large area to popularize and use.
This detection method detect to as if the nucleic acid of SV5 virus, compare with other method that detects antiviral antibody, greater advantage is arranged aspect the sample (like animal derived biological products) that does not have antiviral antibody detecting.
In sum, have bigger practical significance in the detection range of the present invention SV5 in animal derived biological products or relevant raw materials and animal, have a extensive future.
Below in conjunction with specific embodiment the present invention is explained further details.
Description of drawings
The RT-PCR detected result of Fig. 1 for the SV5 cell culture being carried out with primer of the present invention
Fig. 2 is the sensitivity analysis result of RT-PCR detection method of the present invention
Fig. 3 is the sensitivity analysis result of RT-PCR detection method of the present invention
Embodiment
Following embodiment only 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 normal condition such as people such as sambrook the condition described in the volume " molecular cloning experiment guide ", or according to the condition of manufacturer's suggestion.The primer is synthetic by Shanghai Ying Jun company, and the work of all sequences mensuration is given birth to the worker by Shanghai and accomplished.
One, is used for SV5 virus is carried out the primer design that RT-PCR detects
M gene (SEQ ID No:3 in the sequence table) according to SV5 is a template design primer, and the PCR product size of this primer is 317bp, and primer sequence is following:
Upstream primer MF:5 '-TGAGTGGCAGGAATACTGGATG-3 ' (SEQ ID No:1 in the sequence table)
Downstream primer MR:5 '-AGCGGCGATGCTTAGATGAAAT-3 ' (SEQ ID No:2 in the sequence table)
Two, the RT-PCR of SV5 virus detects
With method of the present invention to SV5 virus seed culture of viruses (numbering: 1～16) carry out RT-PCR and detect, concrete grammar may further comprise the steps:
1, the extraction of RNA in the cell culture of SV5 infection
1.1 get the frozen nutrient solution 0.5mL of SV5, add the ice-cold Trizol Reagent (available from Invitrogen) of 0.5mL, concussion mixing 10s, room temperature is placed 10min.
1.2 add the 0.2mL chloroform, concuss 2～3min, room temperature leaves standstill 10min, 4 ℃, the centrifugal 15min of 12000rpm.
1.3 draw supernatant in new centrifuge tube, add the ice-cold Virahol of equal-volume, mixing, room temperature leaves standstill 10min, 4 ℃, the centrifugal 15min of 12000rpm.
1.4 abandon supernatant, with 75% ethanol (precooling is in-20 ℃) 1mL washing RNA deposition once after, 4 ℃, the centrifugal 7min of 8000rpm.
1.5 abandon supernatant, be inverted drying at room temperature 10min.
Remove Rnase water dissolution RNA 1.6 add 20 μ l, 55～60 ℃ of water-bath hydrotropy 10min.
Measure the RNA concentration of being extracted with the uv-spectrophotometric appearance, through measuring, the RNA concentration of being extracted is 0.2095mg/mL.
2, the rt of SV5 viral RNA
The SV5 viral RNA that obtains with step 1 is synthetic its cDNA of template rt, and the PCR reaction system (does not add following reagent) as follows successively in 0.2mL has the PCR thin-walled tube of Rnase:
Remove the dH of Rnase
2O 7 μ l
5×buffer(Promega?AMV 5μl
Reverse?Transcriptasse，
catalog#?M5108)
DNTP (concentration 2.5mM) 4 μ l
Random primer (concentration 0.5 μ l
500μg/mL)
RNA (concentration 0.25 μ g/ μ l) 8 μ l
AMV (concentration 10u/ μ l) 0.5 μ l
Amount to 25 μ l
The reverse transcription reaction condition is: 37 ℃ of 90min, 72 ℃ of 15min, 4 ℃ of 5min.
3, the pcr amplification of SV5 seed culture of viruses
With step 2 synthetic cDNA is template, under the guiding of primer MF and MR, carries out pcr amplification, and the PCR reaction system (adds following reagent) as follows successively in 0.2mL PCR thin-walled tube:
10×buffer(TaKaRa?Taq
TM?Hot?2μl
Start?Version?DR007A)
DNTP (concentration 2.5mM) 1.6 μ l
dH
2O 12.9μl
Upstream primer (concentration 10 μ M) 1 μ l
Downstream primer (concentration 10 μ M) 1 μ l
CDNA (concentration 50ng/ μ l) 1 μ l
Hs Taq enzyme (concentration 5u/ μ l) 0.5 μ l
Amount to 20 μ l
The PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 30s then, 58 ℃ of 30s, 72 ℃ of 1min, totally 35 circulations; Last 72 ℃ of 5min.
4, the result observes
After reaction finishes, get 5 μ l pcr amplification products and carry out 2% agarose gel electrophoresis and detect (damping fluid is TAE, 100 volts 30 minutes), observation and write down the result under gel imaging system.
Result (M:250bp Marker as shown in Figure 1; The RT-PCR product of 1～16:SV5 virus seed culture of viruses); The seed culture of viruses of different batches, SV5 content is different, and the content that has is seldom even do not have; Seed culture of viruses 1～5,7～9,11,14～15 has all detected M gene (the purpose band of the down visible 317bp of uv lamp); The PCR product is served the Hai Shenggong order-checking, and the sequence on result and the NCBI (the SEQ ID No:4 in NC 002578 sequence table) is compared, and coincidence rate is 99%.
The sensitivity analysis of the RT-PCR detection method of test one, SV5 of the present invention virus
The SV5 viral RNA that embodiment 1 step 2 obtains is done 10 respectively
-1～10
-9Serial dilution carries out the RT-PCR amplification respectively with method of the present invention.
Detected result (M:250bp Marker as shown in Figure 2; The 1:SV5 virus stock solution used; 2～10: 10
-1～10
-9Dilution), visible this method has higher sensitivity, and the maximum dilution multiple that can detect is 10
-4, corresponding RNA concentration is 0.21ng/ μ l.
The specificity analyses of the RT-PCR detection method of test two, SV5 of the present invention virus
Use with several kinds of nearer viruses of SV5 sibship and do contrast: Sendai virus (SV), lymphocytic choriomeningitis virus (LCM), CDV (CDV), mumps virus (EPV) and Measles virus (MV); Carry out RT-PCR simultaneously with method of the present invention and detect, reaction system and reaction conditions are identical with embodiment 1.
Detected result (M:100bp Marker as shown in Figure 3; 1～7:SV5, Sendai virus (SeV), lymphocyte choroid plexus encephalitis (LCM), dog infective virus (CDV), mumps virus (EPV), Measles virus (MV), Vero cell); Have the purpose band that the SV5 virus amplification has gone out 317bp only; And other seed culture of viruses is not all seen this specific band, explains that the RT-PCR detection method of SV5 virus of the present invention has excellent specificity.
The RT-PCR detection kit of embodiment 2, SV5 virus
To be used for SV5 virus is carried out primer M1 (10 μ M) 100 μ l that RT-PCR detects and M2 (10 μ M) 100 μ l, Invitrogen TRIzol cat log15596-018 50mL, Promega AMV ReverseTranscriptasse, catalog#M5108, TaKaRa Taq
TMHot Start Version DR007A, SV5DNA10 μ l, DEPC water 1mL, dd water 1mL pack jointly, obtain the RT-PCR detection kit of SV5 virus.
Claims (2)
1. be used for SV5 virus is carried out the primer that RT-PCR detects; Be that M gene section nucleic acid with SV5 designs for detected object; The upstream primer of said primer is represented that by the nucleotide sequence of SEQ ID NO:1 in the sequence table downstream primer is represented by the nucleotide sequence of SEQ ID NO:2 in the sequence table.
2. one kind is used for SV5 virus is carried out the test kit that RT-PCR detects, and comprises the said primer that is used for SV5 virus is carried out the RT-PCR detection of claim 1.
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