CN103509877A - Fluorescence quantitative PCR kit used for detecting PRV, and application thereof - Google Patents
Fluorescence quantitative PCR kit used for detecting PRV, and application thereof Download PDFInfo
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Abstract
The invention relates to a fluorescence quantitative PCR rapid detection kit used for specifically detecting pig pseudorabies virus (PRV) infection, and an application thereof. The kit comprises a specific primer pair used for amplifying PRV gE gene conserved region, and a specific TaqMan fluorescent-labeled probe. An upstream primer sequence of the specific primer pair is 5'-GAGGACGACGGGCTGTAC-3', and a downstream primer sequence of the specific primer pair is 5'-GGACATCAACAGGCGGTTG-3'. The sequence of the TaqMan probe is 5'-TGGGTCCATTCGTCACTTCCG-3'. The 5' end of the TaqMan probe is labeled with a fluorescent reporter group FAM. The 3' end of the TaqMan probe is labeled with a fluorescence quenching group BHQ1 which is not intrinsically fluorescent. The kit can be used for rapid qualitative and quantitative detections of PRV infection, and can be used in identification detections of PRV gE gene-deleted vaccine strains and/or PRV wild strains.
Description
Technical field
The invention belongs to viral nucleic acid detection field, be specifically related to quantitative fluorescent PCR quick detection kit and application thereof that a kind of specific detection porcine pseudorabies virus (PRV) infects.
Background technology
Pseudoabies (Pseudorabies, PR) be called again AujeszkyShi disease, by herpetoviridae Alphaherpesviridae pseudorabies virus (Pseudorabies Virus, PRV) a kind of acute infectious disease causing, wherein, the multiple domestic animals such as pig, ox, sheep and wildlife are this viral main infection object, and the infection symptoms such as performance is generated heat, very itched, encephalomyelitis.Pseudoabies was reported in Hungary early than 1902, was widely current since then in all over the world.China since nineteen forty-seven this disease of reported first, up to now, in succession 20Duo Ge province come into vogue this disease (Li Sulian. the thinking [J] to Pseudorabies Gene-deleted Vaccine strain research. Sichuan animal and veterinary, 2001,3 (28): 27-29.).Pig is main host and the contagium of pseudorabies virus, healthy negative pig with sick pig, be with malicious pig directly to contact all can to infect this disease.Pregnant sow infects pseudorabies virus can cause that miscarriage, mummy tire, stillborn foetus and product are weak young conventionally; It is nervous symptoms that sucking piglets infects pseudorabies virus multiple, is suppurative encephalitis; It is Respiratory symptoms that child care pig and growing and fattening pigs are infected pseudorabies virus multiple; Adult Pig infects pseudorabies virus and mostly is inapparent infection; 2 week age, the death rate of the onset with interior Infection in Piglets pseudorabies virus can reach 100%.At present, pseudorabies virus infects has become one of major disease of serious harm pig industry, and has caused huge financial loss to global aquaculture especially pig industry.
At present, the method that is usually used in detecting PRV comprises activation and detection technique, tissue block cultivation and Coculture techniques, nucleic acid hybridization technique and the round pcr etc. of latent virus in Serologic test, body.The defect such as that wherein, serological test method exists is time-consuming, effort, accuracy rate are low; Tissue block cultivate and Coculture techniques in virus separation there is sensitivity, the advantage such as special, but there are grow (approximately 3 weeks) consuming time, the defects such as complex operation.Although molecular biological ordinary method can make up the deficiency of aforementioned detection method in some aspects, there is the not high defect of false positive, environmental pollution, circulation ratio, thereby limited it, apply.In recent years, emerging Real-Time Fluorescent Quantitative PCR Technique is on the basis of regular-PCR technology, in amplification reaction system, add a pair of Auele Specific Primer and specific fluorescent probe or fluorescence dye, utilization can Real-Time Monitoring fluorescent PCR instrument detect pathogenic agent target nucleotide sequences, this technology has not only overcome the shortcoming that normal PCR technology exists, and it is reliable to have visual result, high specificity, highly sensitive, the quick advantage such as simple, existence from molecular biology level detection to viral nucleic acid intuitively, become the important technology of diagnosis and detection disease clinically.And, Real-Time Fluorescent Quantitative PCR Technique is detecting aspect pseudoabies for detection of PRV infected pigs and latent infection pig, can also quick test distinguish the wild poison of porcine pseudorabies virus and vaccine virus, the infective dose that even can detection by quantitative infects PRV (ten thousand surpasses, Wen Guoyuan, Pan Zi book etc. rapid detection Pseudorabies virus fluorescent quantitative PCR technique is set up and application [J]. Chinese virusology, 2006,21 (5): 485-489.; Zhao Li, Cui Baoan, Chen Hongying etc. differentiate the foundation [J] of the wild poison of pseudorabies virus and vaccine virus fluorescence quantifying PCR method. biotechnology journal, 2008,24 (7): 1149-1154.).
Real-Time Fluorescent Quantitative PCR Technique comprises two kinds of dye method and probe methods, wherein, dye method is to utilize the dyestuff (as SYBR Green) that can be combined with double-stranded DNA to be attached to double-stranded DNA product inside, according to the fluorescent signal detecting, realize again the object of detection by quantitative, but have the defect that cannot distinguish primer dimer and non-specific product signal; Probe method is on conventional PCR basis, to add a specific fluorescent probe (as TaqMan probe), according to the FRET (fluorescence resonance energy transfer) principle producing between the report fluorophor of probe both sides mark and fluorescent quenching group, the fluorescence signal intensity of release and the proportional relation of the amount of amplified production and realize detection by quantitative.
The superiority of Real-Time Fluorescent Quantitative PCR Technique purifies PRV tool to level in Chinese large-sized intensive industrialized piggery and is of great significance.But, lab assistant is when being used Real-Time Fluorescent Quantitative PCR Technique, conventionally need to buy respectively fluorescence dye and enzyme system, and spend the plenty of time for designing the work such as suitable primer probe and optimizing reaction system, and in current clinical detection or research practice, still do not develop a kind of reagent kit product that can realize rapid detection PRV by simple operations, and all there is the problem that can not meet reality needs in the aspect such as the accuracy of real-time quantitative PCR detection technique, specificity, sensitivity and circulation ratio.
Summary of the invention
The PCR kit for fluorescence quantitative that the object of the present invention is to provide a kind of specific detection porcine pseudorabies virus to infect, it is characterized in that, described test kit comprise for the Auele Specific Primer of the porcine pseudorabies virus gE gene conservative region that increases to specific TaqMan fluorescence labeling probe, wherein, the right upstream primer sequence of described Auele Specific Primer is 5 '-GAGGACGACGGGCTGTAC-3 ' (SEQ ID NO:1), the right downstream primer sequence of Auele Specific Primer is 5 '-GGACATCAACAGGCGGTTG-3 ' (SEQ ID NO:2), the sequence of described TaqMan probe is 5 '-TGGGTCCATTCGTCACTTCCG-3 ' (SEQ ID NO:3), and 5 ' end mark fluorescent reporter group FAM of TaqMan probe, the non-luminous fluorescent quenching group B of 3 ' end mark HQ1 itself, the right quantity of Auele Specific Primer of porcine pseudorabies virus gE gene conservative region of being preferred for increasing is at least one pair of, more preferably the quantity of specific TaqMan fluorescence labeling probe is at least one.
In the preferred technical solution of the present invention, described Auele Specific Primer is to being included in PCR reaction solution with specific TaqMan probe, wherein, described PCR reaction solution is comprised of dNTP mixture, the TaqMan probe of 0.1 μ mol/L, the downstream primer of the upstream primer of 0.2 μ mol/L, 0.2 μ mol/L and the appropriate aseptic double-distilled water of 1 * PCR Buffer, 0.5mmol/L.
In the preferred technical solution of the present invention, described test kit also comprises arbitrary component or its combination of DNA extraction reagent, Taq archaeal dna polymerase, positive quality control product and negative quality control product.
In the preferred technical solution of the present invention, described positive quality control product is the T vector plasmid that contains porcine pseudorabies virus gE gene conserved sequence.
The main technical principle of the quantitative fluorescent PCR that specific detection porcine pseudorabies virus of the present invention infects: Real-Time Fluorescent Quantitative PCR Technique is by adding fluorophor in PCR reaction system, utilizes the variation of fluorescent signal to detect in real time the variation of product amount in pcr amplification reaction.
The object of the present invention is to provide PCR kit for fluorescence quantitative of the present invention for the application of the method for fast qualitative detection by quantitative porcine pseudorabies virus, be preferred for differentiate detecting any or its combination of PRV gE gene-deleted vaccine strain HePRV street strain.
The object of the present invention is to provide PCR kit for fluorescence quantitative of the present invention for detection of the application whether existing in sample to be checked in the method for porcine pseudorabies virus DNA.
The object of the present invention is to provide PCR kit for fluorescence quantitative of the present invention for the application of the method for fast qualitative detection by quantitative porcine pseudorabies virus, be preferred for the infection of fast qualitative detection by quantitative porcine pseudorabies virus or porcine pseudorabies virus and infect early stage any or its combination, more preferably for detection by quantitative porcine pseudorabies virus copy number.
The object of the present invention is to provide a kind of method of utilizing fluorescent quantitative PCR technique specific detection porcine pseudorabies virus (PRV) to infect, comprise the steps:
1) extract the viral DNA in testing sample;
2) utilize PCR kit for fluorescence quantitative detecting step 1 of the present invention) viral DNA amount in the testing sample that obtains, obtain.
In the preferred technical solution of the present invention, utilize the result of the method for fluorescent quantitative PCR technique qualitative detection porcine pseudorabies virus (PRV) infection to judge as follows: 1) sample to be tested Ct value≤35, and curve has obvious Exponential growth stage, and measurement result is effective, report sample to be tested is positive; 2) during sample to be tested 35 < Ct value < 40, if the Ct value repeating once is still less than 40, and curve has obvious Exponential growth stage, and report sample to be tested is positive; 3) the Ct value or the Ct value that can't detect sample to be tested are 40, the DNA content < 10 of PRV
1copies/ μ L(is lower than detecting lower limit), report sample to be tested is negative.
In the preferred technical solution of the present invention, the method of utilizing fluorescent quantitative PCR technique detection by quantitative porcine pseudorabies virus (PRV) to infect comprises the steps:, according to the Ct value of the positive plasmid standards for quantitation of the PRV of each sample to be tested and known initial copy number, to calculate the initial copy number of sample to be tested.
In order clearly to explain protection scope of the present invention, the present invention defines as follows to term:
Positive quality control product of the present invention is to utilize the method for gene clone to insert the T vector plasmid of one section of porcine pseudorabies virus gE gene conserved sequence, and its insertion sequence is SEQ ID NO:4.
Negative quality control product of the present invention is the diluent of health pig serum.
Ct value (cycle threshold of the present invention, when the fluorescent signal that Ct) refers to amplified production in pcr amplification process reaches the threshold value of setting the amplification cycles number of times of process, there is linear relationship in the logarithm of the initial copy number of Ct value and template, template DNA amount is more, the cycle number that fluorescence arrives threshold value is fewer, and Ct value is less.Utilize the standard substance of known initial copy number can make typical curve, then according to the Ct value that detects the testing sample obtaining, can be calculated by typical curve the initial copy number of testing sample.
Except as otherwise noted, while the present invention relates to the per-cent between liquid and liquid, described per-cent is volume/volume per-cent; While the present invention relates to the per-cent between liquid and solid, described per-cent is volume/weight per-cent; While the present invention relates to the per-cent between solid and liquid, described per-cent is weight/volume percent; All the other are weight/weight percent.
Compared with prior art, the present invention has following useful technique effect:
1. the present invention has optimized the composition of the PCR kit for fluorescence quantitative of specific detection porcine pseudorabies virus (PRV) infection, comprise optimized for the Auele Specific Primer of the porcine pseudorabies virus gE gene conservative region that increases to specific TaqMan fluorescence labeling probe, also optimized and comprised that Auele Specific Primer forms PCR reaction solution with specific TaqMan probe, described test kit has that the quantitative accuracy of detection is high, specificity is good, false positive rate is low, highly sensitive, detect the advantages such as quick.
2. under the detection of the PCR kit for fluorescence quantitative that specific detection porcine pseudorabies virus of the present invention (PRV) infects, be limited to 10
1the virus quantity of Copies/ μ L, has advantages of that detection sensitivity is high.
3. 5 ' end mark fluorescent reporter group FAM of TaqMan probe used in the fluorescent quantitative PCR technique that specific detection porcine pseudorabies virus of the present invention (PRV) infects, 3 ' end mark non-luminous fluorescent quenching group B HQ1 itself, reduced background fluorescence signal, improve signal to noise ratio, significantly improved the accuracy of experimental result.
4. the fluorescent quantitative PCR technique that specific detection porcine pseudorabies virus of the present invention (PRV) infects all carries out in whole amplification and testing process in same sealed tube, the false positive of effectively having avoided Aerosol Pollution and having caused, has easy and simple to handle, automatization, (whole testing process only needs 1-1.5 hour), the advantage such as accurate fast.
5,, immunofluorescence separated with virus compared with technology such as conventional PCR, the fluorescent quantitative PCR technique that specific detection porcine pseudorabies virus of the present invention (PRV) infects has significantly improved detection efficiency, and has easy and simple to handle, automatization, (whole testing process only needs 1-1.5 hour), the advantage such as accurate fast.
Accompanying drawing explanation
The amplification curve of the 1st group of primer and probe in Fig. 1 embodiment 1, wherein, A, B, C, D are respectively dilution 10 times, 100 times, 1000 times, the 10000 Bei porcine pseudorabies virus DNA of street strain, E is that pig parvoviral DNA, F are that pig gyrate virus II type DNA, G are that porcine pseudorabies virus gE gene-deleted vaccine strain DNA, H are water contrasts, related description in Fig. 2-Fig. 6 is identical therewith, claims again " lower same ".
The amplification curve of the 2nd group of primer and probe in Fig. 2 embodiment 1.
The amplification curve of the 3rd group of primer and probe in Fig. 3 embodiment 1.
The amplification curve of the 4th group of primer and probe in Fig. 4 embodiment 1.
The amplification curve of the 5th group of primer and probe in Fig. 5 embodiment 1.
The amplification curve of the 6th group of primer and probe in Fig. 6 embodiment 1.
The specific kinetic curve of fluorescent quantitative PCR technique that Fig. 7 specific detection porcine pseudorabies virus of the present invention (PRV) infects, wherein, A is that positive quality control product, B are that porcine pseudorabies virus street strain, C are that pig parvoviral, D are that pig gyrate virus II type, E are that the strain of porcine pseudorabies virus gE gene-deleted vaccine, F are negative quality control products.
The kinetic curve of the fluorescent quantitative PCR technique sensitivity that Fig. 8 specific detection porcine pseudorabies virus of the present invention (PRV) infects, wherein, A-F is respectively 10
6, 10
5, 10
4, 10
3, 10
2, 10
1the detection kinetic curve of copy number, G is negative quality control product.
The typical curve of the fluorescent quantitative PCR technique sensitivity that Fig. 9 specific detection porcine pseudorabies virus of the present invention (PRV) infects.
Embodiment
For the ease of understanding the present invention, especially exemplified by following examples.Its effect should be understood to be further explanation of the present invention and explanation, but not for limiting the scope of the invention.
embodiment 1the screening of porcine pseudorabies virus (PRV) fluorescence quantitative PCR detection primer and probe
1, the design of primer and probe
Gene order with reference to China PRV announcing in Genebank, and combined with fluorescent PCR primer probe design feature, utilize Primer Express and DNAStar software design for primer and the probe of PRV conserved regions fragment, multipair primer and the probe (in Table 1) of design are carried out to shaker test, by document (Zhao Li, Cui Baoan, Chen Hongying etc., TaqMan real-time fluorescence quantitative PCR detects foundation and the Preliminary Applications [J] of porcine pseudorabies virus method. Chinese Preventive Veterinary Medicine report, 2009, 31(2): the primer of detection PRV of report and probe sequence 137-140.) (in table 1 the 6th group) compare research in contrast, therefrom filter out the good primer of specificity and susceptibility and probe.
Primer and the probe sequence of table 1 amplification PRV
Note: the PRpf in table 1 represents upstream primer, and PRpr represents downstream primer, and PRpb represents probe.
2, the shaker test of primer and probe
Under identical condition, extract pig parvoviral (PPV) DNA, pig gyrate virus II type (PCV-II) the DNA, PRV DNA of street strain and PRV gE gene-deleted vaccine strain DNA; The DNA of PRV street strain of extraction is carried out to 10 times of dilutions and form 4 gradients, dilute 10 times, 100 times, 1000 times, 10000 times.With PCR, react premixed liquid and (be purchased Premix Ex Taq
tMtAKARA) 5 synthetic cover primer probes of his-and-hers watches 1 design carry out shaker test, and with the primer of bibliographical information and probe (the 6th group) in contrast, therefrom choose that amplification efficiency is high, specificity and the good primer of susceptibility and probe be as test kit of the present invention primer used and probe.The results are shown in Figure 1-Fig. 6, wherein, the A in Fig. 1-Fig. 6, B, C, D are respectively dilution 10 times, 100 times, 1000 times, the 10000 Bei PRV DNA of street strain, and E is that PPV DNA, F are that PCV-II DNA, G are that PRV gE gene-deleted vaccine strain DNA, H are water contrasts.
From Fig. 1-Fig. 6, the amplification curve of the 4th group of primer and probe is level and smooth, susceptibility and specificity are better, and amplification efficiency is high, for this reason, preferably the primer of the 4th group and probe sequence be as primer and the probe of test kit of the present invention, i.e. upstream primer (PRpf): 5 '-GAGGACGACGGGCTGTAC-3 '; Downstream primer (PRpr): 5 '-GGACATCAACAGGCGGTTG-3 '; Fluorescent probe (PRpb): 5 ' FAM-TGGGTCCATTCGTCACTTCCG-BHQ13 '.
embodiment 2the composition of porcine pseudorabies virus fluorescent quantificationally PCR detecting kit and optimization
The optimization of primer concentration and probe concentration: the in the situation that other components being constant in reaction system, the right concentration of Auele Specific Primer is selected respectively 0.1 μ mol/L, 0.2 μ mol/L, 0.3 μ mol/L, 0.4 μ mol/L, 0.5 μ mol/L, the concentration of specific probe is selected respectively 0.05 μ mol/L, 0.1 μ mol/L, 0.2 μ mol/L, 0.3 μ mol/L, 0.4 μ mol/L, carry out PCR preliminary experiment, preferably Auele Specific Primer is to being 0.2 μ mol/L, and specific probe concentration is 0.1 μ mol/L.
The optimization of dNTP mixture concentration: the in the situation that other components being constant in reaction system, the concentration of dNTP mixture is selected respectively 0.1mmol/L, 0.2mmol/L, 0.3mmol/L, 0.4mmol/L, 0.5mmol/L, 0.6mmol/L, carry out PCR, preferably dNTP mixture concentration is 0.5mmol/L.
The optimization of Taq archaeal dna polymerase consumption: the in the situation that other components being constant in reaction system, the concentration of Taq archaeal dna polymerase is selected respectively 1.5U/ reaction, 2U/ reaction, 2.5U/ reaction, 3U/ reaction, 3.5U/ reaction, carry out PCR, preferably Taq archaeal dna polymerase consumption is 2.5U/ reaction.
The optimization of loop parameter: according to target nucleotide length and Taq DNA polymerase activity, annealing temperature and extension time are optimized, the loop parameter of optimization is: 95 ℃ of denaturations 3 minutes (min); Then, 95 ℃ 5 seconds (sec), 60 ℃ of 40sec(collect fluorescent signal), 40 circulations (cycles).
By screening and the optimization of preliminary experiment, determine the composed as follows of test kit:
1.PRV-PCR reaction solution: each reaction system comprises 10 * PCRBuffer(damping fluid) 2.5 μ L, 25mmol/LdNTP mixture 0.5 μ L, 10 μ mol/L TaqMan probe 0.25 μ L, 10 μ mol/L upstream primer 0.5 μ L, 10 μ mol/L downstream primer 0.5 μ L, aseptic double-distilled water (DEPC) 17.25 μ L, totally 21.5 μ L.
2. viral DNA extracting solution: be purchased (the raw work in Shanghai is produced), 30mL/ bottle.
3.DEPC water: 1mL/ pipe.
4.DNA polysaccharase: Taq archaeal dna polymerase (5U/ μ L), 25 μ L/ pipes.
5. positive quality control product: the T vector plasmid that contains porcine pseudorabies virus gE gene conserved sequence (SEQ ID NO:4), 100 μ l/ pipes.
6. negative quality control product: health pig serum, packing after PBS dilution, 300 μ L/ pipes.
embodiment 3the application of porcine pseudorabies virus quantitative fluorescent PCR quick detection kit
The extraction of 1 viral DNA
The extraction of viral DNA in 1.1 testing samples (tissue-type): get sample to be tested 50-100mg, be placed in aseptic 1.5ml centrifuge tube, the ratio that is 1:5 in the weightmeasurement ratio of tissue sample: PBS adds PBS liquid, the abundant homogenate of Syrup-homogenizing instrument, the centrifugal 10min of 4000rpm, get supernatant liquor 200 μ L, be placed in new 1.5mL centrifuge tube aseptic, that pollute without DNA enzyme, number standby.
Get the 1.5mL centrifuge tube that without DNA enzyme pollute suitable with testing sample quantity, perform mark.Respectively add 600 μ LDNA extracting solutions, then add respectively corresponding sample to be tested supernatant liquor and each 200 μ L of negative quality control product, after vibration 30sec mixes, room temperature is placed 10min; Again vibrate after 30sec, add 700 μ L Virahols, vibration mixes, the centrifugal 15min of 12,000rpm; Move and abandon supernatant, note not touching the DNA precipitation at the pipe end, add 1000 μ L 70% ethanol, after the vibration several seconds, the centrifugal 5min of 12,000rpm room temperature; Move and abandon supernatant, do not touch pipe end precipitation DNA, again the of short duration centrifugal several seconds, careful suction abandoned residual supernatant; Add 200 μ L DEPC water, with pipettor, carefully blow and beat the membranaceous throw out on the pipe end and tube wall, make its dissolving; Flick and mix, it is directly carried out to PCR experiment, or standby below being kept at-20 ℃.
The extraction of 1.2 testing samples (liquid-type) DNA: directly measure 200 μ L testing samples, be placed in 1.5ml centrifuge tube aseptic, that pollute without DNA enzyme, and establish negative control (health pig serum dilution 200 μ L), to test accordingly centrifuge tube and perform mark, add respectively again 600 μ L DNA extraction liquid, after vibration 30sec mixes, room temperature is placed 10min; Again vibrate after 30sec, add 700 μ L Virahols, vibration mixes, the centrifugal 15min of 12,000rpm; Move and abandon supernatant, note not touching the DNA precipitation at the pipe end, add 1000 μ L 70% ethanol, after the vibration several seconds, the centrifugal 5min of 12,000rpm room temperature; Move and abandon supernatant, do not touch pipe end precipitation DNA, again the of short duration centrifugal several seconds, move and abandon residual supernatant; Add 200 μ L DEPC water, with pipettor, carefully blow and beat the membranaceous throw out on the pipe end and tube wall, make its dissolving; Flick and mix, it is directly carried out to PCR experiment, or standby below being kept at-20 ℃, and wherein, described liquid-type testing sample is selected from serum, celiolymph, nasopharynx washing lotion, cells and supernatant etc.
2, pcr amplification
The preparation of 2.1PCR reaction system
The pipe number of setting the required PCR reaction tubes of pcr amplification is n, wherein, n=1 manages negative quality control product+6 pipe PRV positive quality control product, and (before use, with DEPC water, by 1 times of positive quality control product gradient dilution, 10 times, 100 times, 1000 times, 10000 times, 100000 times, obtaining concentration is 1 * 10
6copies/ μ L, 1 * 10
5copies/ μ L, 1 * 10
4copies/ μ L, 1 * 10
3copies/ μ L, 1 * 10
2copies/ μ L, 1 * 10
1the plasmid standards for quantitation series of copies/ μ L) the pipe number of+sample to be tested.
Before each PCR reaction, need to prepare premixed liquid, the consisting of of described premixed liquid, the Taq enzyme of the PRV PCR reaction solution of 21.5 μ L and 0.5 μ L.Take out PRV-PCR reaction solution, room temperature is melted and is put upside down and mixes, the centrifugal 10sec of 2000rpm; Take out Taq enzyme, the centrifugal 10sec of 2000rpm, collects the enzyme storage liquid that is bonded at tube wall; Measure the aequum of each reagent in premixed liquid, added in the centrifuge tube of proper volume, completely put upside down and mix, the centrifugal 10sec of 2000rpm, obtains.
In n the PCR reaction tubes of setting, respectively add after the above-mentioned premixed liquid of 22 μ L, in the PCR reaction tubes of setting, add each 3 μ L of the sample to be tested DNA of extraction, negative quality control product and positive quality control product respectively again, cover tightly pipe lid, be placed in fluorescent PCR instrument (StepOne Plus, ABI) upper, that records sample to be tested puts position, hole.
The setting of 2.2PCR reaction parameter
The first step: 95 ℃, 3 minutes (min), 1 circulation (cycle);
Second step: 95 ℃, 5 seconds (sec), and 60 ℃, 40sec(collects fluorescent signal), 40cycles; Wherein, need suitably adjust reaction parameter according to the detecting instrument using.
3, result is judged
Interpretation of result condition is set: click assay surface, get the fluorescent signal of 3-10 circulation or 3-15 circulation and determine baseline (baseline).Threshold value (threshold) setting principle is the vertex just above the amplification curve (random noise line) of normal negative quality control product and negative sample with threshold line, does not occur Ct value and intersects and be as the criterion with the exponential phase of positive quality control product.
Result is judged: there is obvious Exponential growth stage 1) sample to be tested Ct value≤35, and curve, and measurement result is effective, and report sample to be tested is positive; 2) during sample to be tested 35 < Ct value < 40, if the Ct value repeating once is still less than 40, and curve has obvious Exponential growth stage, and report sample to be tested is positive; 3) the Ct value or the Ct value that can't detect sample to be tested are 40, the DNA content < 10 of PRV
1copies/ μ L(is lower than detecting lower limit), report sample to be tested is negative.
4, sample to be tested is quantitative
According to the Ct value of the positive plasmid standards for quantitation of the PRV of each sample to be tested and known initial copy number, instrument automatic analysis calculates the initial copy number of sample to be tested.
5, the specific test of test kit of the present invention
Under identical condition, extract pig parvoviral (PPV) DNA, pig gyrate virus II type (PCV-II) DNA and PRV gE gene-deleted vaccine strain DNA, utilize porcine pseudorabies virus quantitative fluorescent PCR quick detection kit of the present invention to detect above-mentioned viral DNA, and establish negative control group (negative quality control product, health pig serum dilution) and positive controls (positive quality control product), to verify the specificity of this test kit.The results are shown in Figure 7, wherein, the A in Fig. 7 is that positive quality control product, B are that the DNA of porcine pseudorabies virus street strain, C are that pig parvoviral DNA, D are that pig gyrate virus II type DNA, E are that porcine pseudorabies virus gE gene-deleted vaccine strain DNA, F are negative quality control products.
As seen from Figure 7, test kit of the present invention can detect porcine pseudorabies virus specifically, wherein, the detected result of porcine pseudorabies virus sample and positive quality control product is positive, and the detected result of pig parvoviral, pig gyrate virus II type, porcine pseudorabies virus gE gene-deleted vaccine strain and negative quality control product is all negative.
6, the sensitivity test of test kit of the present invention
PRV positive quality control product (1 * 10
1-1 * 10
6copies/ μ L) as template, utilize porcine pseudorabies virus quantitative fluorescent PCR quick detection kit of the present invention to detect, the results are shown in Figure 8, wherein, the A-F in Fig. 8 is respectively 10
6copies/ μ L, 10
5copies/ μ L, 10
4copies/ μ L, 10
3copies/ μ L, 10
2copies/ μ L, 10
1the detection kinetic curve of the PRV positive quality control product copy number of Copies/ μ L, G is negative quality control product.
As seen from Figure 8, the fluorescent quantitative PCR technique that specific detection porcine pseudorabies virus of the present invention (PRV) infects is 10
1copies/ μ L-10
6in the scope of Copies/ μ L, obtain good kinetic curve, and its detectability is low to moderate 10
1the virus quantity of copies/ μ L, has good detection sensitivity.And the fluorescent quantitative PCR technique that specific detection porcine pseudorabies virus of the present invention (PRV) infects is 10
1copies/ μ L-10
6typical curve in the scope of Copies/ μ L is desirable, the results are shown in Figure 9.
As seen from Figure 9, the linearly dependent coefficient (R of typical curve of the present invention
2) more than 0.99, thering is error less, confidence level is compared with advantages of higher.
7, replica test
The PCR kit for fluorescence quantitative that utilizes specific detection porcine pseudorabies virus of the present invention (PRV) to infect detects the testing sample of 3 groups of different virus content, study its repeatability, gained detected result is learned after processing by statistics, and the variation coefficient in calculating group (C.V), the results are shown in Table 2.
Repeatability between table 2 sample sets detects
From table 2, the C.V value of the detected result of the PCR kit for fluorescence quantitative that specific detection porcine pseudorabies virus of the present invention (PRV) infects is all less than 3%, illustrates that difference is not remarkable, has good repeatability.
embodiment 4the detection effect research of porcine pseudorabies virus (PRV) quantitative fluorescent PCR quick detection kit
The present embodiment comparative studies porcine pseudorabies virus quantitative fluorescent PCR of the present invention quick detection kit and the detected result of isolation of virus to porcine pseudorabies virus, result has confirmed that the coincidence rate of porcine pseudorabies virus quantitative fluorescent PCR quick detection kit of the present invention and Virus Isolation method is higher, and can specificly distinguish porcine pseudorabies virus gE gene-deleted vaccine Zhu He porcine pseudorabies virus street strain.
(1) testing sample is divided into two groups, adopts respectively porcine pseudorabies virus quantitative fluorescent PCR quick detection kit of the present invention (being called for short quantitative fluorescent PCR group) and Virus Isolation method (being called for short Virus Isolation group) to be detected.
Testing sample amounts to 30 parts, and 15 parts every group, wherein, 5 parts of every group is pseudorabies living vaccines (production of Bartha-K61 Zhu， Wuhan Chopper Biology Co., Ltd.), and 10 parts of every group is the nasopharynx washing lotion of doubtful infection Pseudorabies virus pig.
Sample to be tested is with after serum free medium dilution, standby.It is 300IU/mL that nasopharynx washing lotion adds penicillin solution to final concentration, add Streptomycin sulphate to its final concentration to be 100 μ g/mL, standby.The PK-15 cell that grows to individual layer, discards growth media, in 10%, 37 ℃ of thermostat container of inoculation testing sample amount for cultivation liquid measure, adsorbs 1h.The inoculation liquid of inclining, adds the DMEM maintenance medium containing 2% new-born calve serum, puts in 37 ℃ of incubators and cultivates.Every day, observation of cell pathology, if typical cytopathic effect (CPE) appears in cell, and reach 80% above pathology time results virus, went down to posterity.As inoculation for the first time does not occur cytopathy blind passage three generations after cell culture freeze thawing should being had to cytopathy person, collect cell bottle, multigelation 3 times, through the centrifugal 10min of 3000rpm, results supernatant liquor ,-70 ℃ save backup.As acellular pathology still, be judged to be pseudorabies virus and detect negative.Separated virus liquid is done further to identify by neutralization test.
(3) porcine pseudorabies virus quantitative fluorescent PCR quick detection kit detection method of the present invention: the preparation of testing sample and inoculation with the detected result of 3, two groups of embodiment in Table 3.
The detected result comparison of table 3 porcine pseudorabies virus PCR kit for fluorescence quantitative and Virus Isolation method
From table 3, by the method for Virus Isolation, detect 15 duplicate samples, detect altogether 13 parts of positive, wherein, 5 parts of pseudorabies vaccine virus (gE gene-deleted vaccine poison) detected result is all positive; Porcine pseudorabies virus quantitative fluorescent PCR quick detection kit of the present invention detects 15 duplicate samples, detects altogether 8 parts of positive, and wherein, 5 parts of pseudorabies vaccine virus (gE gene-deleted vaccine poison) detected result is all negative.Two kinds of methods are consistent to the detected result of nasopharynx washing lotion.Result of study shows, this test kit can specificly be distinguished porcine pseudorabies virus gE gene-deleted vaccine poison and wild virus infection.
Claims (10)
1. the quantitative fluorescent PCR quick detection kit that a specific detection porcine pseudorabies virus (PRV) infects, it is characterized in that, described test kit comprise for the Auele Specific Primer of the porcine pseudorabies virus gE gene conservative region that increases to specific TaqMan fluorescence labeling probe, wherein, the right upstream primer sequence of described Auele Specific Primer is 5 '-GAGGACGACGGGCTGTAC-3 ', the right downstream primer sequence of Auele Specific Primer is 5 '-GGACATCAACAGGCGGTTG-3 ', the sequence of described TaqMan probe is 5 '-TGGGTCCATTCGTCACTTCCG-3 ', and 5 ' end mark fluorescent reporter group FAM of TaqMan probe, the non-luminous fluorescent quenching group B of 3 ' end mark HQ1 itself, the right quantity of Auele Specific Primer of porcine pseudorabies virus gE gene conservative region of being preferred for increasing is at least one pair of, more preferably the quantity of specific TaqMan fluorescence labeling probe is at least one.
2. quantitative fluorescent PCR quick detection kit as claimed in claim 1, it is characterized in that, described Auele Specific Primer is to being included in PCR reaction solution with specific TaqMan probe, wherein, described PCR reaction solution is comprised of dNTP mixture, the TaqMan probe of 0.1 μ mol/L, the downstream primer of the upstream primer of 0.2 μ mol/L, 0.2 μ mol/L and the appropriate aseptic double-distilled water of 1 * PCR Buffer, 0.5mmol/L.
3. quantitative fluorescent PCR quick detection kit as claimed in claim 1 or 2, described test kit also comprises arbitrary component or its combination of DNA extraction reagent, Taq archaeal dna polymerase, positive quality control product and negative quality control product.
4. the quantitative fluorescent PCR quick detection kit as described in claim 1-3 any one, is characterized in that, described positive quality control product is the T vector plasmid that contains porcine pseudorabies virus gE gene conserved sequence.
5. the quantitative fluorescent PCR quick detection kit of claim 1-4 any one is for the application of the method for fast qualitative/detection by quantitative porcine pseudorabies virus, is preferred for differentiating any or its combination that detects porcine pseudorabies virus gE gene-deleted vaccine strain and porcine pseudorabies virus street strain.
6. the quantitative fluorescent PCR quick detection kit of claim 1-4 any one is for detection of the application whether existing in sample to be checked in the method for porcine pseudorabies virus DNA.
7. the quantitative fluorescent PCR quick detection kit of claim 1-4 any one is for the application of the method for fast qualitative/detection by quantitative porcine pseudorabies virus, be preferred for the infection of fast qualitative/detection by quantitative porcine pseudorabies virus or porcine pseudorabies virus and infect early stage any or its combination, more preferably for detection by quantitative porcine pseudorabies virus copy number.
8. a method of utilizing fluorescent quantitative PCR technique specific detection porcine pseudorabies virus (PRV) to infect, comprises the steps:
1) extract the viral DNA in testing sample;
2) utilize the PCR kit for fluorescence quantitative detecting step 1 described in claim 1-4 any one) viral DNA amount in the testing sample that obtains, obtain.
9. method according to claim 8, utilize the result of the method for fluorescent quantitative PCR technique qualitative detection porcine pseudorabies virus (PRV) infection to judge as follows: 1) sample to be tested Ct value≤35, and curve has obvious Exponential growth stage, and measurement result is effective, report sample to be tested is positive; 2) during sample to be tested 35 < Ct value < 40, if the Ct value repeating once is still less than 40, and curve has obvious Exponential growth stage, and report sample to be tested is positive; 3) the Ct value or the Ct value that can't detect sample to be tested are 40, the DNA content < 10 of PRV
1copies/ μ L, report sample to be tested is negative.
10. method according to claim 8, the method of utilizing fluorescent quantitative PCR technique detection by quantitative porcine pseudorabies virus (PRV) to infect comprises the steps:, according to the Ct value of the positive plasmid standards for quantitation of the PRV of each sample to be tested and known initial copy number, to calculate the initial copy number of sample to be tested.
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| CN103952500A (en) * | 2014-05-21 | 2014-07-30 | 广东温氏食品集团股份有限公司 | Fluorescence quantitative PCR (polymerase chain reaction) detection primers, probe and kit for porcine pseudorabies virus (PRV) wild strains |
| CN105886663B (en) * | 2016-04-26 | 2020-03-27 | 广东省农业科学院动物卫生研究所 | Locked nucleic acid sensitization detection kit for fluorescent quantitative PCR of wild strain of porcine pseudorabies virus |
| CN105886663A (en) * | 2016-04-26 | 2016-08-24 | 广东省农业科学院动物卫生研究所 | Locked nucleic acid sensitivity-enhanced fluorescent quantitative PCR (polymerase chain reaction) detection reagent kit for wild strains of porcine pseudorabies viruses |
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