CN108342510A - The multiple RT-PCR kit and its detection method that BTV-11 types, 17 types, 20 types, 23 types, 24 type genotypings differentiate - Google Patents

The multiple RT-PCR kit and its detection method that BTV-11 types, 17 types, 20 types, 23 types, 24 type genotypings differentiate Download PDF

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CN108342510A
CN108342510A CN201810246410.2A CN201810246410A CN108342510A CN 108342510 A CN108342510 A CN 108342510A CN 201810246410 A CN201810246410 A CN 201810246410A CN 108342510 A CN108342510 A CN 108342510A
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CN108342510B (en
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聂福平
杨俊�
王国民
王昱
黄秋华
李贤良
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Chongqing customs Technology Center
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Inspection & Quarantine Technology Center Of Chongqing Entry-Exit Inspection & Quarantine Bureau
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Abstract

The invention discloses multiple RT PCR kits and its detection method that a kind of 11 types of BTV, 17 types, 20 types, 23 types, 24 type genotypings differentiate, differentiate 11 type of detection blue tongue virus, 17 types, 20 types, 23 types, 24 types simultaneously for single tube.This method devises 5 pairs of PCR specific primers according to various virus VP 2 gene sequence conservation, in addition the universal primer of a pair of of non-biological origin of GeXP principles synthesis is used for reference, universal primer is added respectively in the upstream and downstream of each pair of specific primer constitutes 5 pairs of specific chimeric primers, reverse transcription is carried out with specific primer, universal primer and specific chimeric primer is finally used to build multiplex PCR.Using the multiplex PCR system and condition of optimization, can single tube differentiate simultaneously one or more in detection 5 kinds of genotype of this blue tongue virus, to the other types of BTV and PPRV, FMDV nucleic acid without specific amplification, minimal detectable concentration can reach pg grades.The method of the present invention high sensitivity, high specificity, section when it is laborsaving and be easy to observe result.

Description

The multiple RT- that BTV-11 types, 17 types, 20 types, 23 types, 24 type genotypings differentiate PCR kit and its detection method
Technical field
The invention belongs to molecular biology for detection and detection reagent field, round pcr fields.Specifically, specific It is related to 11 type of blue tongue virus (BTV), 17 types, 20 types, 23 types, 24 type genotype multiple RT-PCR detection kits and detection Method.
Background technology
Blue tongue disease (Bluetongue, BT) is by Reoviridae Orbivirus member's blue tongue virus Caused by (Bluetongue virus, BTV), using insect as a kind of untouchable infectious disease of the ruminant of communication media, Widely distributed, the report of blue tongue disease all occurs for each continent in addition to the Antarctic Continent.BTV can infect most of ruminants, with silk floss Sheep is most susceptible, and shows typical clinical symptoms, and ox and goat are mostly subclinical infection, without manifest symptom, but can long-term band poison, Wild animal and camel can also infect the disease.Due to sick sheep fall ill it is dead, or even if not dead, but its production performance degradation (if the production meat output of milk declines, fetal anomaly, wool destruction, lamb development are bad etc.), huge economic loss is caused, and serious International trade is influenced, OIE is classified as legal notification epidemic disease, and China is classified as a kind of zoonosis.BTV serotype is many It is more, it is verified that have 27, named respectively with BTV1-27, intersecting protective is low between each serotype, the feelings of mixed infection Condition also occurs often, therefore also necessary to Viral typing.Detection method applicable at present mainly has virus to be separately cultured, agar Sugared immunodiffusion, serum neutralization test (VNT), ELISA, Ag-capture ELISA, RT-PCR, qRT-PCR and genetic chip Technology.In addition to VNT cannot to Viral typing, though and VNT can parting, time-consuming, and cost is excessively high, and to the blood for detection Clear quality requirement is high, is not suitable for conventional detection.Domestic and foreign scholars have done correlative study to the parting of virus, but can only single tube pair one Type Viral typing, single tube high throughput have not been reported the method for many types of Viral typing.BTV-11/17/20/23/24 is more A country is found, and BTV-11/17 betides the U.S. for a long time, and BTV-20,23 are mainly in Australia, and BTV-20 belongs to the big profit of Australia Sub- predominant serotype.In this five kinds of serotypes, China existing BTV-11,23,24 occur.There are cattle and sheep in China with countries in the world And products thereof trade, it is extremely important to the detection of blue tongue disease, be transferred into and out with pre- anti-virus.
Multiplex PCR is a kind of a plurality of specific primer of utilization in same PCR reaction systems while amplifying multiple nucleic acid The method of segment has easy to operate, quick, specific good and sensibility height etc. a little.Traditional multiplex PCR, which can usually exist, to be drawn Vie each other between object it is big and the problem of limit its flux and sensitivity, Gexp-PCR universal primers and the end of upstream and downstream 5 ' difference Specific chimeric primer with universal primer upstream and downstream carries out PCR amplification, and in amplification, universal primer is incorporated in specificity The both ends of chimeric primers, it is former wheel by low concentration specific chimeric primer dominate amplification, behind by high concentration universal primer Leading amplification, can reduce the competition between each special primer, increase the detection flux and detection sensitivity of single tube PCR.It uses for reference Gexp-PCR universal primer principles, but unlike it adds fluorescent dye on universal primer, make PCR product need not be special It could analyze, can be analyzed on other inexpensive instruments, such multiplex PCR can increase single tube detection flux in Gexp-PCR And sensitivity, and cost can be reduced.
Invention content
The object of the present invention is to provide a kind of high sensitivity, high specificity, section when it is laborsaving and be easy to observe result it is more Weight RT-PCR kit and detection method differentiate detection blue tongue virus (BTV) 11 type, 17 types, 20 types, 23 for single tube simultaneously Type, 24 types.
The present invention adopts the following technical scheme that achieve the goals above：BTV-11 types, 17 types, 20 types, 23 types, 24 types exist Specific amplification is carried out in same RT-PCR reaction systems.
BTV-11 types, 17 types, 20 types, 23 types, 24 type genotypings differentiate multiple RT-PCR kit, including various anti- Transcription primers pipe, Mix PCR reaction solutions pipe, positive control pipe, negative control pipe and sterile deionized water pipe.
The various reverse transcription primer pipe：
BTV-11 reverse transcription primer pipes：DNA sequence dna is the BTV-11 downstream primers 0.5OD of SEQ ID NO.2；
BTV-17 reverse transcription primer pipes：DNA sequence dna is the BTV-17 downstream primers 0.5OD of SEQ ID NO.4；
BTV-20 reverse transcription primer pipes：DNA sequence dna is the BTV-20 downstream primers 0.5OD of SEQ ID NO.6；
BTV-23 reverse transcription primer pipes：DNA sequence dna is the BTV-23 downstream primers 0.5OD of SEQ ID NO.8；
BTV-24 reverse transcription primer pipes：DNA sequence dna is the BTV-24 downstream primers 0.5OD of SEQ ID NO.10；
The Mix PCR reaction solutions pipe is made of following reaction solution：
DNA sequence dna is the 0.75 μ L of general sense primer of 100 μm of ol/L of SEQ ID NO.11；
DNA sequence dna is the 0.75 μ L of general reverse primer of 100 μm of ol/L of SEQ ID NO.12；
DNA sequence dna is the 0.375 μ L of BTV-11 specific chimerics sense primer of 10 μm of ol/L of SEQ ID NO.13；
DNA sequence dna is the 0.375 μ L of BTV-11 specific chimerics downstream primer of 10 μm of ol/L of SEQ ID NO.14；
DNA sequence dna is the 0.625 μ L of BTV-17 specific chimerics sense primer of 10 μm of ol/L of SEQ ID NO.15；
DNA sequence dna is the 0.625 μ L of BTV-17 specific chimerics downstream primer of 10 μm of ol/L of SEQ ID NO.16；
DNA sequence dna is the 0.25 μ L of BTV-20 specific chimerics sense primer of 10 μm of ol/L of SEQ ID NO.17；
DNA sequence dna is the 0.25 μ L of BTV-20 specific chimerics downstream primer of 10 μm of ol/L of SEQ ID NO.18；
DNA sequence dna is the 0.25 μ L of BTV-23 specific chimerics sense primer of 10 μm of ol/L of SEQ ID NO.19；
DNA sequence dna is the 0.25 μ L of BTV-23 specific chimerics downstream primer of 10 μm of ol/L of SEQ ID NO.20；
DNA sequence dna is the 0.25 μ L of BTV-20 specific chimerics sense primer of 10 μm of ol/L of SEQ ID NO.21；
DNA sequence dna is the 0.25 μ L of BTV-20 specific chimerics downstream primer of 10 μm of ol/L of SEQ ID NO.22；
12.5 μ L of 2X Premix Taq buffer solutions；
2.5 μ L of sterile deionized water；
Total 20 μ L, for the dosage of single reaction.
The positive control pipe：It is mixed for BTV-11, BTV-17, BTV-20, BTV-23, BTV-24 positive recombinant plasmid in pipe Close object.Positive recombinant plasmid is obtained by following steps：Respectively with following DNA sequence dna primer sets：SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, routinely RT-PCR amplifications, respectively carry PCR purpose products and PMD19-T Body is attached, and expands culture extraction plasmid after being transformed into DH5 α, and PCR identifications, sequencing and NCBI-BLAST are compared and obtained.
The negative control pipe：It is the beef muscle group of no BTV-11, BTV-17, BTV-20, BTV-23, BTV-24 in pipe Knit DNA sample.
The sterile deionized water pipe：1000μL.
BTV-11 types, 17 types, 20 types, 23 types, 24 type multiple RT-PCR detection methods are carried out with the kit, step is such as Under：
(1) prepared by sample to be tested cDNA templates：Sample to be tested full genome is extracted according to commercialization viral RNA extracts kit Group prepares the various cDNA of sample to be tested, takes respectively with various reverse transcription primer and commercialization reverse transcription reagent box operation instructions Equivalent mixing be placed on -20 DEG C it is spare.
(2) PCR amplification system：20 μ L Mix PCR reaction solutions, 5 μ L sample cDNA templates or positive control are negative right According to reaction total volume is 25 μ L.
(3) pcr amplification reaction condition：95℃5min；95 DEG C of 30s, 57 DEG C of annealing 30s, 72 DEG C of 30S, 10 cycles；95℃ 30s, 50 DEG C of 30s, 72 DEG C of 30S, 25 cycles；72 DEG C of 5min, 4 DEG C of preservations.
(4) result judgement：After Qesp100 carries out Capillary Electrophoresis, it is respectively BTV- that 5 absorption peaks, which occurs, in positive control 11 between 172-186bp, and BTV-17 is between 221-239bp, and BTV-20 is between 317-343bp, and BTV-23 is in 377-409bp Between, BTV-24 is between 266-288bp；Negative control is without absorption peak；Sample occurs in the identical position in positive control position Absorption peak is then determined as that the genotype positive, no absorption peak appearance are then determined as genotype feminine gender；Positive quality control does not occur mesh Band or negative control there is band, then result is invalid.
The principle of the present invention is：For the type VP2 genes of BTV-11,17,20,23,24 conserved sequence design high specificity, The similar specific primer each other without complementary or complementary very little of annealing temperature, uses for reference Gexp-PCR correlations again on this basis Principle, the universal primer upstream and downstream composition specificity for adding same a pair of of non-biological origin respectively in the upstream and downstream of specific primer are embedding Primer is closed, finally carries out PCR amplification with the specific chimeric primer of low concentration and the universal primer of high concentration.In PCR amplification When, universal primer is incorporated in the both ends of specific chimeric primer, and the specific chimeric primer of former wheel low concentrations is leading to be expanded, after Amplification is dominated in face by the universal primer of high concentration, can reduce the competition between each special primer, and the detection for overcoming normal PCR is logical Measure low and detection sensitivity difference problem.In the genome of BTV, VP3, VP7 etc. are highly conserved, smaller, the VP2 bases of VP5 variations It is maximum because making a variation between different shaped, it is type specific antigen, but there is also up in the VP2 of the same type virus in different regions 30% variability, therefore in design primer, both the VP2 gene orders of different shaped were compared, also to multiple regional homotypes The VP2 gene orders of BTV are compared, and design multipair primer to be selected and verify one by one, finally select suitable primer.
Multiplex PCR is a kind of a plurality of specific primer of utilization in a PCR reaction systems while amplifying multiple nucleic acid pieces The method of section, has many advantages, such as that easy to operate, quick, specific and sensibility is high, is widely used in animal pathogenic diagnosis. It is cumbersome to avoid conventional method (pathological anatomy, pathogen separation, serological method etc.), time-consuming and laborious, sensibility is low etc. asks Topic.
It is an advantage of the invention that (1) single tube flux is higher, single tube can differentiate 5 kinds of serotypes of detection simultaneously, time saving, laborsaving； (2) quickly, efficiently：Detection time is in 4h or so；(3) high specific：To in addition to BTV11 types, 17 types, 20 types, 23 types, 24 types its Its BTV genotype and poxvirus, foot and mouth disease virus are without amplification；(4) high sensitivity：Minimal detectable concentration be BTV-11,17,20, 23 types are 4.0 × 103Copies/ μ L, BTV-24 type 4.0 × 102copies/μL；(5) reduce pollution：This method PCR product By capillary electrophoresis analysis, the chemical contamination problem of agarose gel electrophoresis can avoid.(6) it compared with Gexp-PCR, is not required to To add fluorescent dye on universal primer, reduce primer cost.
Description of the drawings
Fig. 1 substances RT-PCR builds result；
Fig. 2 multiple RT-PCR temperature optimization results；In figure, the temperature in A~F is respectively：A-55 DEG C, B-56 DEG C, C-57 ℃,D-58℃,E-59℃,F-60℃；
Fig. 3 specific test results；In figure, M2.Size Marker 20-1000bp；1~6. template is followed successively by BTV- 11,17,20,23,24 hybrid template, BTV-11, BTV-17, BTV-20, BTV-23, BTV-24；7~16. templates are followed successively by BTV-1~BTV10；17~21. templates are followed successively by BTV-12~BTV-16；22.BTV-18；23.BTV-19；24.BTV-21； 25.BTV-22；26.BTV-25；27.PRRSV；28.FMDV；29. negative control；
Fig. 4 sensitivity tests result results；In figure, M2.Size Marker 20-1000bp；1~9 respectively 4.0 × 108~100Copies/ μ L plasmid templates；10. negative control；
Fig. 5 repetitive test results；In figure, M.DL2000DNA molecular mass standards；1~9 is respectively 4.0 × 108~ 100Copies/ μ L plasmid templates；10. negative control.
Specific implementation mode
The specific embodiment technical solution that the present invention is further explained is provided below, but the application of the technology of the present invention is not limited to Following embodiment.
Embodiment 1, the design synthesis of primer
According to BTV11 types, 17 types, 20 types, 23 types, the VP2 gene order profits of 24 types virus announced on the websites Genbank The specific primer that this 5 type virus is designed with 5.0 biosoftwares of Primer Premier is respectively labeled as SEQ ID NO.1- SEQ ID NO.10；Gexp-PCR universal primers are used for reference, SEQ ID NO.11, SEQ ID NO.12 are labeled as；Respectively in each spy 5 ' ends of anisotropic upstream and downstream primer constitute specific chimeric primer plus universal primer upstream and downstream, are labeled as SEQ ID NO.13- SEQ ID NO.22, all primers are sent to Hua Da gene chemical synthesis.Specific primer sequence is as follows：
BTV-11 specific forward primer SEQ ID NO.1：5’-CGGTTGCGAATAACTCAT-3’；
BTV-11 specific downstream primer SEQ ID NO.2：5’-ATTGTATACGCATGTCGA-3’；
BTV-17 specific forward primer SEQ ID NO.3：5’-GAGTTCGTCATTCCAGTC-3’；
BTV-17 specific downstream primer SEQ ID NO.4：5’-TCGCTGGTTTATAATTCA-3’；
BTV-20 specific forward primer SEQ ID NO.5：5’-AAATCGTGAGCGAAGTGG-3’；
BTV-20 specific downstream primer SEQ ID NO.6：5’-GGCAGCAATGGCAATGAG-3’；
BTV-23 specific forward primer SEQ ID NO.7：5’-GGCGACAACATGGCTTAC-3’；
BTV-23 specific downstream primer SEQ ID NO.8：5’-GGGTGCGTTATAGGAACA-3’；
BTV-24 specific forward primer SEQ ID NO.9：5’-ACACGAGCAGTTACGAGA-3’；
BTV-24 specific downstream primer SEQ ID NO.10：5’-ATCACTGGGACAGACCAC-3’；
Universal primer SEQ ID NO.11：5’-AGGTGACACTATAGAATA-3’；
Universal primer SEQ ID NO.12：5’-GTACGACTCACTATAGGGAT-3’；
BTV-11 specific chimeric sense primer SEQ ID NO.13：
5’-AGGTGACACTATAGAATACGGTTGCGAATAACTCAT-3’；
BTV-11 specific chimeric downstream primer SEQ ID NO.14：
5’-GTACGACTCACTATAGGGAATTGTATACGCATGTCGA-3’；
BTV-17 specific chimeric sense primer SEQ ID NO.15：
5’-AGGTGACACTATAGAATAGAGTTCGTCATTCCAGTC-3’；
BTV-17 specific chimeric downstream primer SEQ ID NO.16：
5’-GTACGACTCACTATAGGGATCGCTGGTTTATAATTCA-3’；
BTV-20 specific chimeric sense primer SEQ ID NO.17：
5’-AGGTGACACTATAGAATAAAATCGTGAGCGAAGTGG-3’；
BTV-20 specific chimeric downstream primer SEQ ID NO.18：
5’-GTACGACTCACTATAGGGAGGCAGCAATGGCAATGAG-3’；
The specific affine sense primer SEQ ID NO.19 of BTV-23：
5’-AGGTGACACTATAGAATAGGCGACAACATGGCTTAC-3’；
BTV-23 specific chimeric downstream primer SEQ ID NO.20：
5’-GTACGACTCACTATAGGGAGGGTGCGTTATAGGAACA-3’；
BTV-24 specific chimeric sense primer SEQ ID NO.21：
5’-AGGTGACACTATAGAATAACACGAGCAGTTACGAGA-3’；
BTV-24 specific chimeric downstream primer SEQ ID NO.22：
5’-GTACGACTCACTATAGGGAATCACTGGGACAGACCAC-3；
Embodiment 2, positive recombinant plasmid structure
Viral RNA is extracted according to TaKaRa Mini BEST Viral RNA/DNA Extraction Kit kits.Point PrimeScript is not pressed with each specific primerTMOne Step RT-PCR kit amplifications；According to OMEGA Gel Eetraction Kit kit specifications recycle target fragment, are connected to PMD19-T carriers and convert to DH5a, increase bacterium Kit AXGYEN AxyPreP Plasmid Minippep kit are pressed afterwards and extract plasmid, and carry out PCR identifications, PCR positive matter Grain is sent to Hua Da gene sequencing, and sequencing result compares in NCBI, and it is correctly positive plasmid to compare.Utilize spectrophotometer pair Positive recombinant plasmid carries out concentration mensuration, and calculates copy number.
3 substance RT-PCR structures of embodiment and optimization
25 μ L substance PCR detection architectures are built by template of above-mentioned positive plasmid：Permix Taq 12.5 μ L, general F and R (25 μm of ol/L) each 1 μ L, specific chimeric F and R (10 μm of ol/L) each 0.25 μ L, 1 μ L of template, aqua sterilisa supply 25 μ L.Reaction Program：94℃5min；94 DEG C of 30s, if 58 DEG C of 30s, 72 DEG C of 30S, 10 cycles；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 30S, 25 Cycle；72 DEG C of 5min, 4 DEG C of preservations.Product carries out capillary electrophoresis analysis.It anneals on this basis to specific chimeric primer warm Degree (55 DEG C, 56 DEG C, 57 DEG C, 58 DEG C, 59 DEG C, 60 DEG C) optimize, then to specific chimeric primer concentration (0.05 μm of ol/L, 0.1 μm of ol/L, 0.15 μm of ol/L, 0.20 μm of ol/L, 0.25 μm of ol/L) optimization.
With reference to figure 1 the results show that each serotype substance amplifies the product being consistent with purpose product size.Each serum Type theoretical product size is BTV-11 179bp, BTV-17 230bp, BTV-20 330bp, BTV-23393bp, BTV24 277bp。
4 multiple RT-PCR of embodiment is built and optimization
Each positive recombinant plasmid is diluted to 4.0 × 108Copies/ μ L, take equivalent mixing, are with the mixture and respectively Simple substance grain is template, and the multiplex PCR system of 25 μ L is built with specific chimeric primer and universal primer：Permix Taq 12.5μ L, general F and R (100 μm of ol/L) each 0.75 μ L, 5 couples of specific chimeric primer F and R (10 μm of ol/L) each 0.25 μ L, template each 1 μ L, aqua sterilisa supply 25 μ L.Reaction condition：95℃5min；95 DEG C of 30s, 58 DEG C of annealing 30s, 72 DEG C of 30S, 10 cycles；95℃ 30s, 50 DEG C of 30s, 72 DEG C of 30S, 25 cycles；72 DEG C of 5min, 4 DEG C of preservations.Capillary electrophoresis analysis is carried out after reaction.Again Comprehensive substance PCR results to specific primer concentration (0.1~0.25 μm of ol/L) and annealing temperature (55 DEG C, 56 DEG C, 57 DEG C, 58 DEG C, 59 DEG C, 60 DEG C) optimize.As a result capillary electrophoresis analysis is carried out.With the specific primer of 5 kinds of serotype after screening is good 5 kinds of viral RNA mixture reverse transcriptions are cDNA by mixture (isoconcentration is isometric), then should by template verification of cDNA mixtures Method.
As a result when BTV-20,23,24 take 0.1 μm of ol/L, BTV-11 that 0.15 μm of ol/L, BTV-17 is taken to take 0.25 μm of ol/L When, 5 kinds of serotype target fragments expand well；For temperature optimization the results show that when temperature is 57 DEG C, amplification is the best (with reference to figure 2).
5 specific test of embodiment
With 5 kinds of viruses and 5 kinds of virus mixtures and the other type type viruses of BTV, PPR virus PPRV, hoof-and-mouth disease The DNA or cDNA of malicious FMDV makees template, and optimizing established method with above-described embodiment 4 is expanded.
It is shown with reference to the result of figure 3：The five weight RT-PCR methods that this method is established only to BTV-11 types, 17 types, 20 types, 23 types, 24 type viral templates have specific amplification, and expand without special the other types of BTV, PPR virus, foot and mouth disease virus Increase.
6 sensitivity tests of embodiment
By concentration 4.0 × 108Five kinds of isometric mixings of positive plasmid of copies/ μ L, then carry out 10 times of doubling dilution groups At 4.0 × 108~1009 concentration gradients of copies/ μ L are expanded using each concentration gradient as template by excellent 4 optimization method of example of applying Increase.
It is shown with reference to the result of figure 4:The minimal detectable concentration of the type of BTV-11,17,20,23 is 4.0 × 103Copies/ μ L, BTV-24 type minimal detectable concentrations are 4.0 × 102copies/μL。
7 repetitive test of embodiment
According to sensitivity tests as a result, choosing the template for 3 low concentrations that can be detected, each template is weighed twice It is multiple, as repeatedly experiment in group；The plasmid of different batches extraction is subjected to primary repeat as between group by sensitivity testing method It repeats to test.
According to sensitivity tests the results show that the minimal detectable concentration of serotype can reach 4.0 × 10 in 53copies/ μ L, therefore take 4.0 × 105L~4.0 × 10 copies/ μ3The template of copies/ μ 3 concentration of L, each template are repeated twice It is repeated as in group, it is as a result consistent with sensitivity test result；Different batches extraction plasmid is taken to carry out primary weight between being repeated as group It is multiple, it is as a result consistent with sensitivity test result, test result is repeated between group with reference to figure 5.
The preparation of 8 reagent of embodiment
Various reverse transcription primer pipe：BTV-11 reverse transcription primer pipes：Sequence is the BTV-11 downstream primers of SEQ ID NO.2 0.5OD；BTV-17 reverse transcription primer pipes：Sequence is the BTV-17 downstream primers 0.5OD of SEQ ID NO.4；BTV-20 reverse transcriptions Primer pipe：Sequence is the BTV-20 downstream primers 0.5OD of SEQ ID NO.6；BTV-23 reverse transcription primer pipes：Sequence is SEQ ID The BTV-23 downstream primers 0.5OD of NO.8；BTV-24 reverse transcription primer pipes：Sequence is that the downstreams BTV-24 of SEQ ID NO.10 are drawn Object 0.5OD.
Mix PCR reaction solution pipes：Each 0.75 μ L of SEQ ID NO.11 and SEQ ID NO.12 of 100 μm of ol/L；10μmol/ Each 0.375 μ L of SEQ ID NO.13 and SEQ ID NO.14 of L；The SEQ ID NO.15 and SEQ ID NO.16 of 10 μm of ol/L are each 0.625μL；Each 0.25 μ L of SEQ ID NO.17~SEQ ID NO.22 of 10 μm of ol/L；2X Premix Taq buffer solutions 12.5μL；2.5 μ L of sterile deionized water；Totally 20 μ L are single reacting dose, and the amount that each kit does 50 reactions for foot is total 1000μL。
Positive control pipe：Positive plasmid is prepared as described in Example 2, and the positive plasmid of various virus is diluted to 108Copies/ μ L, take isometric mixing, and often pipe fills 260 μ L.
Negative control pipe：The beef muscle without BTV-11, BTV-17, BTV-20, BTV-23, BTV-24 is extracted with kit Tissue DNA sample is diluted to a concentration of 15~20ng/ μ L with sterile deionized water, and often pipe fills 260 μ L.
Sterile deionized water pipe：Often 1000 μ L of pipe.
The assembling of 9 kit of embodiment
Such as the prepared various reverse transcription primer pipe of embodiment 8, Mix PCR reaction solutions pipe, positive control pipe, negative control Pipe, each 1 of sterile deionized water pipe, stick date of manufacture, the term of validity and Product labelling, Cord blood and transport.
SEQUENCE LISTING
<110>Chongqing Entry-Exit Inspection and Quarantine Bureau inspection and quarantine technique center
<120>The multiple RT-PCR kit that BTV-11 types, 17 types, 20 types, 23 types, 24 type genotypings differentiate And its detection method
<160> 22
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.1
cggttgcgaa taactcat 18
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.2
attgtatacg catgtcga 18
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.3
gagttcgtca ttccagtc 18
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.4
tcgctggttt ataattca 18
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.5
aaatcgtgag cgaagtgg 18
<210> 6
<211> 18
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.6
ggcagcaatg gcaatgag 18
<210> 7
<211> 18
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.7
ggcgacaaca tggcttac 18
<210> 8
<211> 18
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.8
gggtgcgtta taggaaca 18
<210> 9
<211> 18
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.9
acacgagcag ttacgaga 18
<210> 10
<211> 18
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.10
atcactggga cagaccac 18
<210> 11
<211> 18
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.11
aggtgacact atagaata 18
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.12
gtacgactca ctatagggat 20
<210> 13
<211> 36
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.13
aggtgacacta tagaatacgg ttgcgaata actcat 36
<210> 14
<211> 37
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.14
gtacgactca ctatagggaa ttgtatacgc atgtcga 37
<210> 15
<211> 36
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.15
aggtgacact atagaataga gttcgtcatt ccagtc 36
<210> 16
<211> 37
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.16
gtacgactca ctatagggat cgctggttta taattca 37
<210> 17
<211> 36
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.17
aggtgacact atagaataaa atcgtgagcg aagtgg 36
<210> 18
<211> 37
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.18
gtacgactca ctatagggag gcagcaatgg caatgag 37
<210> 19
<211> 36
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.19
aggtgacact atagaatagg cgacaacatg gcttac 36
<210> 20
<211> 37
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.20
gtacgactca ctatagggag ggtgcgttat aggaaca 37
<210> 21
<211> 36
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.21
aggtgacact atagaataac acgagcagtt acgaga 36
<210> 22
<211> 37
<212> DNA
<213>Artificial sequence
<400> SEQ ID NO.22
gtacgactca ctatagggaa tcactgggac agaccac 37

Claims (4)

  1. The multiple RT-PCR kit that 1.BTV-11 types, 17 types, 20 types, 23 types, 24 type genotypings differentiate, it is characterized in that： Including various reverse transcription primer pipe, Mix PCR reaction solutions pipe, positive control pipe, negative control pipe and sterile deionized water pipe；
    The various reverse transcription primer pipe is as follows：
    BTV-11 reverse transcription primer pipes：DNA sequence dna is the BTV-11 downstream primers 0.5OD of SEQ ID NO.2；
    BTV-17 reverse transcription primer pipes：DNA sequence dna is the BTV-17 downstream primers 0.5OD of SEQ ID NO.4；
    BTV-20 reverse transcription primer pipes：DNA sequence dna is the BTV-20 downstream primers 0.5OD of SEQ ID NO.6；
    BTV-23 reverse transcription primer pipes：DNA sequence dna is the BTV-23 downstream primers 0.5OD of SEQ ID NO.8；
    BTV-24 reverse transcription primer pipes：DNA sequence dna is the BTV-24 downstream primers 0.5OD of SEQ ID NO.10；
    The Mix PCR reaction solutions pipe is made of following reaction solution：
    DNA sequence dna is the 0.75 μ L of general sense primer of 100 μm of ol/L of SEQ ID NO.11；
    DNA sequence dna is the 0.75 μ L of general reverse primer of 100 μm of ol/L of SEQ ID NO.12；
    DNA sequence dna is the 0.375 μ L of BTV-11 specific chimerics sense primer of 10 μm of ol/L of SEQ ID NO.13；
    DNA sequence dna is the 0.375 μ L of BTV-11 specific chimerics downstream primer of 10 μm of ol/L of SEQ ID NO.14；
    DNA sequence dna is the 0.625 μ L of BTV-17 specific chimerics sense primer of 10 μm of ol/L of SEQ ID NO.15；
    DNA sequence dna is the 0.625 μ L of BTV-17 specific chimerics downstream primer of 10 μm of ol/L of SEQ ID NO.16；
    DNA sequence dna is the 0.25 μ L of BTV-20 specific chimerics sense primer of 10 μm of ol/L of SEQ ID NO.17；
    DNA sequence dna is the 0.25 μ L of BTV-20 specific chimerics downstream primer of 10 μm of ol/L of SEQ ID NO.18；
    DNA sequence dna is the 0.25 μ L of BTV-23 specific chimerics sense primer of 10 μm of ol/L of SEQ ID NO.19；
    DNA sequence dna is the 0.25 μ L of BTV-23 specific chimerics downstream primer of 10 μm of ol/L of SEQ ID NO.20；
    DNA sequence dna is the 0.25 μ L of BTV-20 specific chimerics sense primer of 10 μm of ol/L of SEQ ID NO.21；
    DNA sequence dna is the 0.25 μ L of BTV-20 specific chimerics downstream primer of 10 μm of ol/L of SEQ ID NO.22；
    12.5 μ L of 2X Premix Taq buffer solutions；
    2.5 μ L of sterile deionized water；
    Total 20 μ L, for the dosage of single reaction；
    The positive control pipe：It is mixed for BTV-11, BTV-17, BTV-20, BTV-21, BTV-23 positive recombinant plasmid in pipe Object；
    The negative control pipe：It is the beef muscle tissue of no BTV-11, BTV-17, BTV-20, BTV-23, BTV-24 in pipe DNA sample；
    The sterile deionized water pipe：1000μL.
  2. 2. the multiple RT-PCR that BTV-11 types, 17 types, 20 types, 23 types, 24 type genotypings differentiate according to claim 1 Kit, it is characterized in that：The positive recombinant plasmid is obtained by following steps：Respectively with following DNA sequence dna primer sets：SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, routinely RT-PCR amplifications, respectively by the production of PCR mesh Object is attached with PMD19-T carriers, expands culture extraction plasmid, and PCR identifications, sequencing and NCBI- after being transformed into DH5 α BLAST, which is compared, to be obtained.
  3. 3. the multiple RT-PCR that BTV-11 types, 17 types, 20 types, 23 types, 24 type genotypings differentiate according to claim 1 Kit, it is characterized in that：BTV-11, BTV-17, BTV-20, BTV-23, BTV-24 carry out spy in the same PCR reaction tubes This 5 kinds of genotype are differentiated and are distinguished according to the size of amplified fragments by specific amplification.
  4. 4. carrying out the more of BTV-11, BTV-17, BTV-20, BTV-23, BTV-24 using the kit of claims 1 or 22 or 3 The detection method of weight RT-PCR non-disease diagnostic purposes：Include the following steps：
    (1) prepared by sample to be tested cDNA templates：Sample to be tested full-length genome is extracted according to commercialization viral RNA extracts kit, Respectively with various reverse transcription primer and commercialization reverse transcription reagent box operation instructions, sample to be tested cDNA is prepared, takes equivalent mixed It is even be placed in -20 DEG C it is spare；
    (2) pcr amplification reaction system：20 μ L Mix PCR reaction solutions, 5 μ L sample to be tested cDNA templates or positive control or feminine gender Control, reaction total volume are 25 μ L；
    (3) pcr amplification reaction condition：95℃5min；95 DEG C of 30s, 57 DEG C of annealing 30s, 72 DEG C of 30s, 10 cycles；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 30S, 25 cycles；72 DEG C of 5min, 4 DEG C of preservations；
    (4) result judgement：After Qesp100 carries out Capillary Electrophoresis, it is respectively that BTV-11 exists that 5 absorption peaks, which occurs, in positive control Between 172-186bp, BTV-17 between 221-239bp, BTV-20 between 317-343bp, BTV-23 377-409bp it Between, BTV-24 is between 266-288bp；Negative control is without absorption peak；Sample is inhaled in the identical position in positive control position It receives peak and is then determined as that the genotype positive, no absorption peak appearance are then determined as genotype feminine gender；Positive quality control does not occur purpose There is band in band or negative control, then result is invalid.
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CN115725749A (en) * 2022-11-17 2023-03-03 广州艾基生物技术有限公司 Mouse source cell STR detection kit, method and application

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