CN108220479A - The multiple linking probe amplification identification reagent box of a variety of pig Sudden Death Syndrome cause of diseases can be detected - Google Patents

The multiple linking probe amplification identification reagent box of a variety of pig Sudden Death Syndrome cause of diseases can be detected Download PDF

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CN108220479A
CN108220479A CN201810012643.6A CN201810012643A CN108220479A CN 108220479 A CN108220479 A CN 108220479A CN 201810012643 A CN201810012643 A CN 201810012643A CN 108220479 A CN108220479 A CN 108220479A
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swine fever
pig
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CN108220479B (en
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宋厚辉
周莹珊
王晓杜
邵春艳
陈琳
章先
程昌勇
杨杨
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Zhejiang A&F University ZAFU
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Abstract

The present invention relates to Measurements for Biotechnique, it is desirable to provide a kind of multiple linking probe amplification identification reagent box that can detect a variety of pig Sudden Death Syndrome cause of diseases.The kit includes：Reverse transcription and pre- amplification primer mixed liquor, probe mixed liquor, MLPA buffer solutions, connection buffer solution A, connection buffer solution B, ligase Ligase 65, PCR reaction mixtures, SALSA polymerases, negative control, positive control.Compared with prior art, the present invention has the characteristics that high-throughput, specific good and high sensitivity.6 kinds of cause of diseases that pig is caused to die suddenly can be detected simultaneously, and 96 samples can be detected simultaneously every time using full-automatic nucleic acids instrument.It is designed by probe, sequence alignment and blast analysis, it is ensured that probe is only combined with target gene.Target fragment of corresponding size is only amplified from corresponding templates when being detected, amplification is had no to other cause of diseases.The step for by increasing RT PCR, is enriched with target gene, the minimum target gene for detecting 1 copy.

Description

The multiple linking probe amplification identification reagent box of a variety of pig Sudden Death Syndrome cause of diseases can be detected
Technical field
The present invention provides one kind for detecting pig breeding and respiratory complication virus, swine fever virus, Nipah virus, non- Continent swine fever virus, C.perfringens and actinobacillus pleuropneumoniae multiple linking probe amplification differentiate detection kit and Primer and probe, it can be achieved that simultaneously detect 6 kinds cause pig die suddenly cause of diseases, belong to inspection and quarantine field.
Background technology
Pig breeds and respiratory syndrome viral (PRRSV), swine fever virus (CSFV), Nipah virus (NiPV), African pig Pestivirus (ASFV), C.perfringens (Clostridium perfringens) and actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae) is the encountered pathogenic for causing pig Sudden Death Syndrome.Although China is not found still African swine fever and pig Buddhist nun pa disease, but surrounding countries of China and Asia have the pathogenetic report of disease, should also be subject to weight Depending on makeing a good technical reserve and monitoring and warning, preventing trouble before it happens.
Due to causing the disease more than one of pig Sudden Death Syndrome, in terms of clinical diagnosis, different bacterium (poison) strain virulence are different, and In the presence of the mixed infection with other cause of diseases so that by clinical symptoms and pathological change be difficult that pathogenesis is made a definite diagnosis.This Outside, it since disease morbidity is rapid, needs to determine pathogenesis in the short time and prevent and treat.Although to causing pig sudden death Factor has corresponding method and strategy, but there is no one way to disposably a variety of diseases that pig is caused to die suddenly is carried out Differentiate.Therefore establish it is a kind of can precisely, detection method that is quick, efficiently distinguishing Different Kinds of Pathogens, prevention for pig Sudden Death Syndrome and Occur most important.
Invention content
The technical problem to be solved by the present invention is to overcome deficiency of the prior art, it is sudden to provide a kind of a variety of pigs of energy detection The multiple linking probe of incruable disease cause of disease expands identification reagent box and detects a variety of pig Sudden Death Syndrome cause of diseases simultaneously using the kit Multiple linking probe amplification detection method.
To solve technical problem, the present invention uses following technical scheme：
A kind of multiple linking probe amplification identification reagent box that can detect a variety of pig Sudden Death Syndrome cause of diseases, the kit packet are provided It includes：
(1) reverse transcription and pre- amplification primer mixed liquor；The mixed liquor includes sequence SEQ ID NO：Use shown in 1~9 In pig breeding and respiratory syndrome virus, swine fever virus, the reverse transcription primer of Nipah virus and African swine fever virus, aerogenesis pod Film clostridium and the pre- amplimer of actinobacillus pleuropneumoniae；
(2) probe mixed liquor；The mixed liquor includes sequence SEQ ID NO：Being bred for detecting pig shown in 10~21 It is put with respiratory syndrome virus, swine fever virus, Nipah virus, African swine fever virus, C.perfringens and pig pleuropneumonia The left side of line bar bacterium and right side probe；
(3) MLPA buffer solutions；
(4) buffer solution A is connected；
(5) buffer solution B is connected；
(6) ligase Ligase-65；
(7) PCR reaction mixtures, the mixed liquor include sequence SEQ ID NO:Universal primer shown in 22 and 23；
(8) SALSA polymerases；
(9) negative control；
(10) positive control.
In the present invention, sequence SEQ ID NO：1 is pig breeding and respiratory syndrome virus reverse transcriptase primer；Sequence SEQID NO：2 be swine fever virus reverse transcription primer；Sequence SEQ ID NO：3 be Nipah virus reverse transcription primer；Sequence SEQ ID NO：4 and SEQ ID NO：5 be the forward and reverse primer that African swine fever virus expands in advance respectively；Sequence SEQ ID NO：6 Hes SEQ ID NO：7 be the forward and reverse primer that C.perfringens expands in advance respectively；Sequence SEQ ID NO：8 and SEQ ID NO：9 be the forward and reverse primer that actinobacillus pleuropneumoniae expands in advance respectively；Sequence SEQ ID NO：10 and SEQ ID NO：11 be respectively the breeding of detection pig and the left side probe of respiratory syndrome virus and right side probe；Sequence SEQ ID NO：12 With SEQ ID NO：13 be respectively the detection left side probe of swine fever virus and right side probe；Sequence SEQ ID NO：14 and SEQ ID NO：15 be respectively the detection left side probe of Nipah virus and right side probe；Sequence SEQ ID NO：16 and SEQ ID NO：17 points The left side probe of African swine fever virus and right side probe Wei not detected；Sequence SEQ ID NO：18 and SEQ ID NO：19 are respectively Detect the left side probe of C.perfringens and right side probe；Sequence SEQ ID NO：20 and SEQ ID NO：21 be respectively to detect The left side probe of actinobacillus pleuropneumoniae and right side probe；Sequence SEQ ID NO：22 and SEQ ID NO：23 be logical respectively With forward and reverse primer；Wherein, sequence SEQ ID NO：11, SEQ ID NO：13, SEQ ID NO：15, SEQ ID NO： 17, SEQ ID NO：19 and SEQ ID NO：21 5 ' ends carry out phosphatizing treatment.
In the present invention, the positive control is the mixture of gene in-vitro transcription RNA and positive recombinant plasmid DNA；Wherein Gene in-vitro transcription RNA refers to pig breeding and the M genes of respiratory syndrome virus, the raq gene and Nipah virus of swine fever virus N genes in-vitro transcription RNA, positive recombinant plasmid DNA refer to the plc of the p27 genes of African swine fever virus, C.perfringens The positive recombinant plasmid DNA of the ApxIVA genes of gene and actinobacillus pleuropneumoniae.
The present invention also provides the multiple linking probe expansions for detecting a variety of pig Sudden Death Syndrome cause of diseases simultaneously using aforementioned agents box Increase detection method, operating process is as described below：
(1) paramagnetic particle method extraction sample DNA/RNA；
Extracts kit is total to the magnetic bead DNA/RNA of TIANGEN companies, uses the grand NP968 Full automatic instrument for extracting nucleic acid in day The DNA and RNA in sample are extracted simultaneously, obtain 200 μ L samples；
(2) RNA reverse transcriptions are expanded into cDNA and in advance
One-step method reverse transcription RT-PCR reactions are carried out according to QIAGEN OneStep Ahead RT-PCR Kit specifications； Prepare 25 μ L reaction systems：10 μ L OneStep Ahead RT-PCR Master Mix, 1 μ L OneStep AheadRT- Mix, 5 μ L DNA or RNA, 5 μ L reverse transcriptions and pre- amplification primer mixed liquor (final concentration of 0.5 μM of every primer), 4 μ L H2O is mended Foot；Reaction condition：50 DEG C of 10min, 95 DEG C of 5min；95 DEG C of 15s, 55 DEG C of 20s, 72 DEG C of 20s, 40 cycles；72℃2min；
(3) MLPA is detected
A, DNA is denaturalized
0.2mL PCR reaction tubes are taken, often pipe adds in 0.5 μ L DNA solutions and 4.5 μ L TE, 98 DEG C of denaturation 5min are down to room 25 DEG C of temperature；
B, probe and sample DNA hybridize
Prepare the probe mixed liquor of 3 μ L mixings：+ 1.5 μ L probe mixed liquors of 1.5 μ L MLPA buffer solutions；By probe mixture It adds in above-mentioned PCR pipe, 95 DEG C of incubation 1min, 60 DEG C of hybridization 1-16h, 54 DEG C incubate；
C, the connection of hybridization probe
Prepare 32 μ L connection enzymatic mixtures：25μL dH2+ 1 μ L connections of+3 μ L connections buffer solution B of O+3 μ L connections buffer solution A Enzyme Ligase-65；PCR instrument temperature is down to 54 DEG C, opens pipe lid, adds in 32 μ L connection enzymatic mixtures, and 54 DEG C incubate 15min, and 98 DEG C heating 5min inactivation ligase, 20 DEG C incubation；
D, the PCR amplification of linking probe
Prepare 10 μ L PCR mixed liquors：7.5μL dH2+ 0.5 μ L SALSA polymerases of O+2 μ L PCR reaction mixtures；It takes Go out PCR pipe, add in 10 μ L PCR mixtures at room temperature；Start PCR reactions, reaction condition is：95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C 60s, 35 cycles；72 DEG C of incubation 20min, are down to 15 DEG C；
E, full-automatic nucleic acids instrument analysis：
Pcr amplification product is analyzed with full-automatic nucleic acids instrument (Qsep100DNA Analyzer)；
(4) result description and judgement
A, quality control standard：
Positive control has specific amplification band at 94bp, 106bp, 116bp, 126bp, 135bp, 144bp；
Negative control is without specific amplification band；
If negative control and positive condition are unsatisfactory for conditions above, this time experiment is considered as invalid；
B, result judges：
It is positive：There is specific amplification band at 94bp, represent that there are pig breedings and respiratory syndrome virus in sample； There is specific amplification band at 106bp, represent that there are swine fever virus in sample；There are specific amplification band, table at 116bp There are Nipah virus in sample sheet；There is specific amplification band at 126bp, represent that there are African swine fever viruses in sample； There is specific amplification band at 135bp, represent that there are C.perfringens in sample；There is specific amplification band at 144bp, Represent that there are actinobacillus pleuropneumoniaes in sample；MLPA amplified productions can be sequenced, further confirmed that；
It is negative：Without specific amplification band, show in sample without pig breeding and respiratory syndrome virus, swine fever virus, Nipah virus, African swine fever virus, C.perfringens and actinobacillus pleuropneumoniae.
Compared with prior art, the beneficial effects of the invention are as follows：
It is 1. high-throughput.6 kinds of cause of diseases that pig is caused to die suddenly can be detected simultaneously, it, every time can be same using full-automatic nucleic acids instrument When detect 96 samples, antidiastole and emergency diagnosis for pathogen provide high-throughput detection technique.
2. specificity is good.It is designed by probe, sequence alignment and blast analysis, it is ensured that probe is only mutually tied with target gene It closes.And clinically common cause of disease is detected, this method only can amplify mesh of corresponding size from its corresponding template Segment, amplification is had no to other cause of diseases.
3. high sensitivity.Common MLPA methods at least need the target dna of 6000 copies.By increase RT-PCR this One step is enriched with target gene, the minimum target gene for detecting 1 copy of the present invention.
Description of the drawings
Fig. 1 is pig breeding and respiratory syndrome virus, swine fever virus, Nipah virus, African swine fever virus, perfringens Clostridium and the multiple linking probe amplification Capillary Electrophoresis peak figure of actinobacillus pleuropneumoniae.
It is shown in figure：Pig breeds and specific amplification peak of the respiratory syndrome virus at 94bp；Swine fever virus exists Specific amplification peak at 106bp；Specific amplification peak of the Nipah virus at 116bp；African swine fever virus is at 126bp Specific amplification peak；Specific amplification peak of the C.perfringens at 135bp；Actinobacillus pleuropneumoniae is at 144bp Specific amplification peak.
Fig. 2 is pig breeding and respiratory syndrome virus, swine fever virus, Nipah virus, African swine fever virus, perfringens Clostridium and the multiple linking probe amplification Capillary Electrophoresis glue figure of actinobacillus pleuropneumoniae.
M：1000bp DNA marker, NC：No template control, Lane1,2,3,4,5,6 respectively with PRRSV, CSFV, The RNA of NiPV in-vitro transcriptions, the recombinant plasmid of ASFV p27 genes, C.perfringens and actinobacillus pleuropneumoniae are mould Plate carries out MLPA detections with mixed probe.Lane 7 carries out MLPA inspections using the mixture of 6 kinds of cause of diseases as template, with mixed probe It surveys.
Fig. 3 is respectively with the RNA of PRRSV, CSFV, NiPV in-vitro transcription, the recombinant plasmid of ASFV p27 genes, aerogenesis pod Film clostridium and actinobacillus pleuropneumoniae are template, and the Capillary Electrophoresis glue figure of MLPA detections is carried out with PRRSV probes.
M：1000bp DNA marker, Lane 1：Using PRRSV as template, Lane 2,3,4,5,6 respectively with CSFV, The RNA of NiPV in-vitro transcriptions, the recombinant plasmid of ASFV p27 genes, C.perfringens and actinobacillus pleuropneumoniae are mould Plate, NC：No template control.
Fig. 4 is respectively with the RNA of PRRSV, CSFV, NiPV in-vitro transcription, the recombinant plasmid of ASFV p27 genes, aerogenesis pod Film clostridium and actinobacillus pleuropneumoniae are template, and the Capillary Electrophoresis glue figure of MLPA detections is carried out with CSFV probes.
M：1000bp DNA marker, Lane 1：Using CSFV as template, Lane 2,3,4,5,6 respectively with PRRSV, The RNA of NiPV in-vitro transcriptions, the recombinant plasmid of ASFV p27 genes, C.perfringens and actinobacillus pleuropneumoniae are mould Plate, NC：No template control.
Fig. 5 is respectively with the RNA of PRRSV, CSFV, NiPV in-vitro transcription, the recombinant plasmid of ASFV p27 genes, aerogenesis pod Film clostridium and actinobacillus pleuropneumoniae are template, and the Capillary Electrophoresis glue figure of MLPA detections is carried out with NiPV probes.
M：1000bp DNA marker, Lane 1：Using the RNA of NiPV in-vitro transcriptions as template, Lane 2,3,4,5,6 point Not using PRRSV, the recombinant plasmid of CSFV, ASFV p27 gene, C.perfringens and actinobacillus pleuropneumoniae as template, NC：No template control.
Fig. 6 is respectively with the RNA of PRRSV, CSFV, NiPV in-vitro transcription, the recombinant plasmid of ASFV p27 genes, aerogenesis pod Film clostridium and actinobacillus pleuropneumoniae are template, and the Capillary Electrophoresis glue figure of MLPA detections is carried out with ASFV probes.
M：1000bp DNA marker, Lane 1：Using the recombinant plasmid of ASFV p27 genes as template, Lane 2,3,4, 5th, 6 respectively using the RNA, C.perfringens and actinobacillus pleuropneumoniae of PRRSV, CSFV, NiPV in-vitro transcription as template, NC：No template control.
Fig. 7 is respectively with the RNA of PRRSV, CSFV, NiPV in-vitro transcription, the recombinant plasmid of ASFV p27 genes, aerogenesis pod Film clostridium and actinobacillus pleuropneumoniae are template, and the Capillary Electrophoresis glue of MLPA detections is carried out with C.perfringens probe Figure.
M：1000bp DNA marker, Lane 1：Using C.perfringens as template, Lane 2,3,4,5,6 respectively with The RNA of PRRSV, CSFV, NiPV in-vitro transcription, the recombinant plasmid of ASFV p27 genes and actinobacillus pleuropneumoniae are mould Plate, NC：No template control.
Fig. 8 is respectively with the RNA of PRRSV, CSFV, NiPV in-vitro transcription, the recombinant plasmid of ASFV p27 genes, aerogenesis pod Film clostridium and actinobacillus pleuropneumoniae are template, and the capillary of MLPA detections is carried out with actinobacillus pleuropneumoniae probe Running gel figure.
M：1000bp DNA marker, Lane 1：Using actinobacillus pleuropneumoniae as template, Lane 2,3,4,5,6 Respectively using the RNA of PRRSV, CSFV and NiPV in-vitro transcription, the recombinant plasmid of ASFV p27 genes and C.perfringens as mould Plate, NC：No template control.
Specific embodiment
Multiplex ligation-dependent probe amplification (multiplex ligation-dependent probe Amplification, MLPA) it is a kind of new skill high-throughput, that qualitative and quantitative analysis is carried out for target sequence in determined nucleic acid Art.The hybridization check of nucleic acid and PCR chain amplifications are combined by the technology, are analyzed so as to fulfill the efficient specificity of target molecule, The target gene different to 45 is detected and quantitative analysis in same reaction tube.The basic principle of MLPA includes probe and target Sequence DNA is hybridized, and later by connection, PCR amplification, product passes through capillary electrophoresis separation and data collection, DNA analysis Software to the data of collection analyze and finally be drawn a conclusion.Each MLPA probes include two oligonucleotide fragments, one by Chemical synthesis, one is prepared by the phage-derived methods of M13；Each probe includes one section of primer sequence and one section of specific sequence Row.In MLPA reactions, two oligonucleotide fragments are all hybridized with target sequence, and connecting two parts using ligase later visits Needle.Coupled reaction high special, only when two probes hybridize completely with target sequence, i.e. target sequence and probe-specific sequence is complete Complete complementary, two sections of probes could be connected into a complete single nucleic acid strands by ligase；, whereas if target sequence and probe sequence It is not fully complementary, even if only there are one the difference of base, it may result in hybridization not exclusively, make coupled reaction that can not carry out.Connection After the completion of reaction, with the probe that connects of a pair of of universal primer amplification, the length of the amplified production of each pair of probe be all it is unique, Range is in 130~480bp.Finally, pass through capillary electrophoresis separation amplified production, software analysis, it was therefore concluded that.
The present invention establishes while detects the high specificity of 6 kinds of pathogen of induction pig Sudden Death Syndrome, susceptibility height and can weigh The good MLPA of renaturation differentiates detection method, and antidiastole and emergency diagnosis for pathogen provide high-throughput detection technique.
The MLPA reactions of one standard at least need the target dna of 6000 copies.To improve the susceptibility of detection cause of disease, The present invention improves it, the step of introducing reverse transcription.By by RNA reverse transcriptions into cDNA, and with PCR pre-expansion gaining marks DNA, to improve Monitoring lower-cut.Primer and probe is designed for the most conservative gene of virus and bacterium, sequence is obtained from GenBank , and sequence alignment is carried out to determine what is guarded the most in each gene with MUSCLE Alignment (Geneious 8.1.4) Region.Primer is designed (sequence is shown in Table 1) with Primer3, is expanded for specific reverse transcription with pre-, these primer amplifications Segment includes the region that MLPA probes combine.Probe design flow (Designing with reference to disclosed in MRC-Holland synthetic MLPA probes,Version 15).Probe is respectively in connection with pig breeding and the M bases of respiratory syndrome virus Cause, the N genes of the raq gene of swine fever virus, Nipah virus, the p27 genes of African swine fever virus, C.perfringens plc bases Because of the region (sequence is shown in Table 2) of the ApxIVA most conservative genes of, actinobacillus pleuropneumoniae, a pair of PCR expansions are finally designed The universal primer of increasing (sequence is shown in Table 3).Left side probe is made of two sections of nucleotide, one section be PCR amplification universal primer, one section It is virus-specific sequence (LHS)；Right side probe is made of two sections of nucleotide, and one section is virus-specific sequence (RHS), one section It is the universal primer of PCR amplification, and 5 ' ends of right side probe carry out phosphatizing treatment.It is distinguished by the probe for designing different length Different Kinds of Pathogens.All primer and probes carry out blast analyses all in ncbi database, to ensure the special of primer and probe Property.Different size of PCR product is obtained by the PCR amplification of template denaturation, probe hybridization, connection and universal primer, by complete Automatic nucleic acids instrument analysis is realized to being detected while 6 kinds of cause of diseases.
1 pig of table breeds and respiratory syndrome virus, swine fever virus, Nipah virus, African swine fever virus, perfringens shuttle Bacterium and actinobacillus pleuropneumoniae reverse transcription and pre- amplification title and sequence
2 pig of table breeds and respiratory syndrome virus, swine fever virus, Nipah virus, African swine fever virus, perfringens shuttle Bacterium and the short probe of actinobacillus pleuropneumoniae and long probe title and sequence
Wherein, above-mentioned SEQ ID NO：11, SEQ ID NO：13, SEQ ID NO：15, SEQ ID NO：17,
SEQ ID NO：19 and SEQ ID NO：21 5 ' ends carry out phosphatizing treatment；
3 universal primer sequence of table
The present invention established using multiplex ligation-dependent probe amplification and meanwhile detect pig breeding and respiratory syndrome it is viral, Swine fever virus, Nipah virus, African swine fever virus, C.perfringens and actinobacillus pleuropneumoniae detection method and group Kit is filled.
Detect pig breeding and respiratory syndrome virus, swine fever virus, Nipah virus, African swine fever virus, perfringens Clostridium and the multiple linking probe amplification detection kit of actinobacillus pleuropneumoniae, it is composed of the following components：
(1) reverse transcription and pre- amplification primer mixed liquor, including pig breeding and respiratory syndrome virus, swine fever virus, Buddhist nun The reverse transcription primer and African swine fever virus of pa virus, the pre- amplification of C.perfringens and actinobacillus pleuropneumoniae are drawn Object, the primer sequence are shown in Table 1, a concentration of 2.5 μM of every primer in mixed liquor；
(2) probe mixed liquor, including the breeding of detection pig and respiratory syndrome virus, swine fever virus, Nipah virus, non- Continent swine fever virus, the left side of C.perfringens and actinobacillus pleuropneumoniae and right side probe, the sequence of the probe are shown in Table 2, wherein, the SEQ ID NO：11, SEQ ID NO：13, SEQ ID NO：15, SEQ ID NO：17, SEQ ID NO：19 With SEQ ID NO：21 5 ' ends carry out phosphatizing treatment；A concentration of 1.33nM of each probe in mixed liquor；
(3) MLPA buffer solutions；
(4) buffer solution A is connected；
(5) buffer solution B is connected；
(6) ligase Ligase-65；
(7) PCR reaction mixtures, including sequence table SEQ ID NO:22 and SEQ ID NO：General shown in 23 draws Object；
(8) SALSA polymerases；
(9) negative control：TE；
(10) positive control：For pig breeding and the M genes of respiratory syndrome virus, the raq gene and Ni Pa of swine fever virus The N gene in-vitro transcription RNA of virus, the plc genes of p27 genes, C.perfringens and pig pleura with African swine fever virus The mixture of the positive recombinant plasmid DNA of the ApxIVA genes of Actinobacillus.
Pig breeds the preparation with respiratory syndrome virus M gene in-vitro transcription RNA：RT-PCR amplification pig breedings and breathing Viral (JX-7 plants) the M genes of trace integration sign simultaneously recycle amplified production, and length 339bp is cloned into pGEM-5zf carriers and (is purchased from Promega companies), positive recombinant plasmid is obtained after sequencing confirms, is named as pGEM-PRRSV-M.Using the plasmid of purifying as mould After plasmid is linearized with MluI enzymes, in-vitro transcription is carried out with the MEGAscript T7 kits of Ambion companies for plate, will In-vitro transcription product is precipitated with LiCl, and the ddH of no RNase is dissolved in after the washing of 70% ethyl alcohol2In O, nucleic acid electrophoresis detection RNA synthesis Integrality and correctness, and measure RNA concentration.Pig breeding and the RNA of respiratory syndrome virus M gene in-vitro transcription are ordered Entitled PRRSV-M-RNA.Pig breeds and SEQ ID NO in JX-7 plants of M gene orders of respiratory syndrome virus such as sequence table：24 It is shown.
The preparation of E 2 gene of Classical Swine Fever in-vitro transcription RNA：RT-PCR expands swine fever virus (SM plants) raq gene and recycles Amplified production, length 345bp are cloned into pGEM-5zf carriers (purchased from Promega companies), are obtained after sequencing confirms positive Recombinant plasmid is named as pGEM-CSFV-E2.Using the plasmid of purifying as template, after plasmid is linearized with MluI enzymes, use The MEGAscript T7 kits of Ambion companies carry out in-vitro transcription, in-vitro transcription product are precipitated with LiCl, 70% ethyl alcohol The ddH of no RNase is dissolved in after washing2In O, the integrality and correctness of nucleic acid electrophoresis detection RNA synthesis, and measure RNA concentration. Pig breeding and the RNA of respiratory syndrome virus M gene in-vitro transcription are named as CSFV-E2-RNA.E 2 gene of Classical Swine Fever SEQ ID NO in sequence such as sequence table：Shown in 25.
The preparation of pig Nipah virus N gene in-vitro transcriptions RNA：Since the country is there has been no Nipah virus, by database Sequence alignment, the sequence for choosing 292bp relatively conservative in Nipah virus N genes carries out outer-gene synthesis, is cloned into PGEM-5zf carriers (are purchased from Promega companies), are named as pGEM-NiPV-N.Using the plasmid of purifying as template, plasmid is used After the linearisation of MluI enzymes, in-vitro transcription is carried out with the MEGAscript T7 kits of Ambion companies, by in-vitro transcription product It is precipitated with LiCl, the ddH of no RNase is dissolved in after the washing of 70% ethyl alcohol2In O, the integrality and just of nucleic acid electrophoresis detection RNA synthesis True property, and measure RNA concentration.The RNA of pig Nipah virus N gene in-vitro transcriptions is named as NiPV-N-RNA.Pig Nipah virus N SEQ ID NO in gene order such as sequence table：Shown in 26.
The preparation of African swine fever virus p27 gene masculine recombinant plasmids：Due to the country, there has been no African swine fever viruses, pass through Sequence alignment in database, the sequence for choosing 278bp relatively conservative in African swine fever virus gene p27 carry out outer-gene Synthesis is cloned into pGEM-5zf carriers (purchased from Promega companies), is named as pGEM-ASFV-p27.African swine fever virus p27 SEQ ID NO in gene order such as sequence table：Shown in 27.
The preparation of C.perfringens plc gene masculine recombinant plasmids：PCR amplification C.perfringens (CVCC1126) Plc genes simultaneously recycle amplified production, length 211bp, pGEM-5zf carriers (purchased from Promega companies) are cloned into, through sequencing Positive recombinant plasmid is obtained after confirmation, is named as pGEM-C.perfringens-plc.C.perfringens pcl gene orders are such as SEQ ID NO in sequence table：Shown in 28.
The preparation of actinobacillus pleuropneumoniae ApxIVA gene masculine recombinant plasmids：PCR amplification pig pleuropneumonia unwrapping wire Bacillus (CVCC3559) ApxIVA genes simultaneously recycle amplified production, and length 223bp is cloned into pGEM-5zf carriers and (is purchased from Promega companies), positive recombinant plasmid is obtained after sequencing confirms, is named as pGEM-A.pleuropneumoniae- ApxIVA.SEQ ID NO in actinobacillus pleuropneumoniae ApxIVA gene orders such as sequence table：Shown in 29.
The present invention provides detect pig breeding and respiratory syndrome virus, swine fever virus, Nipah virus, African pig simultaneously The multiple linking probe amplification detection method of pestivirus, C.perfringens and actinobacillus pleuropneumoniae, concrete operations stream Journey is as follows：
1. paramagnetic particle method extracts sample DNA/RNA；
Extracts kit is total to the magnetic bead DNA/RNA of TIANGEN companies, uses the grand NP968 Full automatic instrument for extracting nucleic acid in day The DNA and RNA in sample are extracted simultaneously, obtain 200 μ L samples.
2.RNA reverse transcriptions amplification into cDNA and in advance
One-step method reverse transcription RT-PCR reactions are carried out according to QIAGEN OneStep Ahead RT-PCR Kit specifications. Prepare 25 μ L reaction systems：10 μ L OneStep Ahead RT-PCR Master Mix, 1 μ L OneStep AheadRT- Mix, 5 μ L DNA or RNA, 5 μ L reverse transcriptions and pre- amplification primer mixed liquor (final concentration of 0.5 μM of every primer), 4 μ L H2O is mended Foot.Reaction condition：50 DEG C of 10min, 95 DEG C of 5min；95 DEG C of 15s, 55 DEG C of 20s, 72 DEG C of 20s, 40 cycles；72℃2min.
3.MLPA is detected
1) DNA is denaturalized
0.2mL PCR reaction tubes are taken, often pipe adds in 0.5 μ L DNA solutions and 4.5 μ L TE, 98 DEG C of denaturation 5min are down to room 25 DEG C of temperature.
2) probe and sample DNA hybridize
Prepare the probe mixed liquor of 3 μ L mixings：+ 1.5 μ L probe mixed liquors of 1.5 μ L MLPA buffer solutions.By probe mixture It adds in above-mentioned PCR pipe, 95 DEG C of incubation 1min, 60 DEG C of hybridization 1-16h, 54 DEG C incubate.
3) connection of hybridization probe
Prepare 32 μ L connection enzymatic mixtures：25μL dH2+ 1 μ L connections of+3 μ L connections buffer solution B of O+3 μ L connections buffer solution A Enzyme Ligase-65.PCR instrument temperature is down to 54 DEG C, opens pipe lid, adds in 32 μ L connection enzymatic mixtures, and 54 DEG C incubate 15min, and 98 DEG C heating 5min inactivation ligase, 20 DEG C incubation.
4) PCR amplification of linking probe
Prepare 10 μ L PCR mixed liquors：7.5μL dH2+ 0.5 μ L SALSA polymerases of O+2 μ L PCR reaction mixtures.It takes Go out PCR pipe, add in 10 μ L PCR mixtures at room temperature.Start PCR reactions, reaction condition is：95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C 60s, 35 cycles；72 DEG C of incubation 20min, are down to 15 DEG C.
5) full-automatic nucleic acids instrument analysis：
Pcr amplification product is analyzed with full-automatic nucleic acids instrument (Qsep100DNA Analyzer).
4. result describes and judgement
1) quality control standard：
Positive control has specific amplification band at 94bp, 106bp, 116bp, 126bp, 135bp, 144bp.
Negative control is without specific amplification band.
If negative control and positive condition are unsatisfactory for conditions above, this time experiment is considered as invalid.
2) result judges：
It is positive：There is specific amplification band at 94bp, represent that there are pig breedings and respiratory syndrome virus in sample； There is specific amplification band at 106bp, represent that there are swine fever virus in sample.There are specific amplification band, table at 116bp There are Nipah virus in sample sheet；There is specific amplification band at 126bp, represent that there are African swine fever viruses in sample； There is specific amplification band at 135bp, represent that there are C.perfringens in sample；There is specific amplification band at 144bp, Represent that there are actinobacillus pleuropneumoniaes in sample.MLPA amplified productions can be sequenced, further confirmed that.
It is negative：Without specific amplification band, show in sample without pig breeding and respiratory syndrome virus, swine fever virus, Nipah virus, African swine fever virus, C.perfringens and actinobacillus pleuropneumoniae.
With reference to specific embodiment, the present invention will be described in detail.
First, the use of kit
The composition of 1 kit
Wherein, MLPA buffer solutions, connection buffer solution A, connection buffer solution B, ligase Ligase-65 and SALSA polymerase Purchased from MRC-Holland companies.
Condition of storage：
1) in addition to positive control, other components please be stored in -15 DEG C to -25 DEG C.Positive control please be stored in -80 DEG C.
2) in order to ensure experiment effect, please within 1 year that receives product using finishing.
2 application methods
2.1 paramagnetic particle methods extract the DNA/RNA of sample；
Extracts kit is total to the magnetic bead DNA/RNA of TIANGEN companies, uses the grand NP968 Full automatic instrument for extracting nucleic acid in day The DNA and RNA in sample are extracted simultaneously, obtain 200 μ L samples.
2.2RNA reverse transcriptions amplification into cDNA and in advance
One-step method reverse transcription RT-PCR reactions are carried out according to QIAGEN OneStep Ahead RT-PCR Kit specifications. Prepare 25 μ L reaction systems：10 μ L OneStep Ahead RT-PCR Master Mix, 1 μ L OneStep AheadRT- Mix, 5 μ L DNA or RNA, 5 μ L reverse transcriptions and pre- amplification primer mixed liquor (final concentration of 0.5 μM of every primer), 4 μ L H2O is mended Foot.Reaction condition：50 DEG C of 10min, 95 DEG C of 5min；95 DEG C of 15s, 55 DEG C of 20s, 72 DEG C of 20s, 40 cycles；72℃2min.
2.3MLPA detection
2.3.1DNA denaturation
0.2mL PCR reaction tubes are taken, often pipe adds in 0.5 μ L DNA solutions and 4.5 μ L TE, 98 DEG C of denaturation 5min are down to room 25 DEG C of temperature.
2.3.2 probe and sample DNA hybridize
Prepare the probe mixed liquor of 3 μ L mixings：+ 1.5 μ L probe mixtures of 1.5 μ L MLPA buffer solutions.By probe mixture It adds in above-mentioned PCR pipe, 95 DEG C of incubation 1min, 60 DEG C of hybridization 1-16h, 54 DEG C incubate.
2.3.3 the connection of hybridization probe
Prepare 32 μ L connection enzymatic mixtures：25μL dH2+ 1 μ L connections of+3 μ L connections buffer solution B of O+3 μ L connections buffer solution A Enzyme Ligase-65.PCR instrument temperature is down to 54 DEG C, opens pipe lid, adds in 32 μ L connection enzymatic mixtures, and 54 DEG C incubate 15min, and 98 DEG C heating 5min inactivation ligase, 20 DEG C incubation.
2.3.4 the PCR amplification of linking probe
Prepare 10 μ L PCR mixed liquors：7.5μL dH2+ 0.5 μ L SALSA polymerases of O+2 μ L PCR reaction mixtures.It takes Go out PCR pipe, add in 10 μ L PCR mixtures at room temperature.Start PCR reactions, reaction condition is：95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C 60s, 35 cycles；72 DEG C of incubation 20min, are down to 15 DEG C.
2.3.5 full-automatic nucleic acids instrument analysis：
Pcr amplification product is analyzed with full-automatic nucleic acids instrument (Qsep100DNA Analyzer).
2.4. result description and judgement
1) quality control standard：
Positive control has specific amplification band at 94bp, 106bp, 116bp, 126bp, 135bp, 144bp.
Negative control is without specific amplification band.
If negative control and positive condition are unsatisfactory for conditions above, this time experiment is considered as invalid.
2) result judges：
It is positive：There is specific amplification band at 94bp, represent that there are pig breedings and respiratory syndrome virus in sample； There is specific amplification band at 106bp, represent that there are swine fever virus in sample.There are specific amplification band, table at 116bp There are Nipah virus in sample sheet；There is specific amplification band at 126bp, represent that there are African swine fever viruses in sample； There is specific amplification band at 135bp, represent that there are C.perfringens in sample；There is specific amplification band at 144bp, Represent that there are actinobacillus pleuropneumoniaes in sample.MLPA amplified productions can be sequenced, carry out validation test.
It is negative：Without specific amplification band, show in sample without pig breeding and respiratory syndrome virus, swine fever virus, Nipah virus, African swine fever virus, C.perfringens and actinobacillus pleuropneumoniae.
Points for attention：
1) DNA sample：
A. the content of salt ion, alcohols etc. in DNA solution is reduced as far as possible.
B. with TE rather than water dissolution or dilution DNA, depurination when avoiding DNA high temperature.
C. by all DNA sample concentration dilutions to roughly equal (it is recommended that 20-40ng/ul) before experiment.
2) probe, ligase Ligase-65, connection buffer solution A should dispense before use, avoid multigelation；
3) it should shake mixing centrifugation before buffer solution and reagent use, gently blow and beat mixing with pipette tips during mixing, pay attention to avoiding It generates bubble or will get on tube wall, all steps containing enzyme can not centrifuge；
4) when plus connecting enzyme reaction, PCR pipe will be placed in PCR instrument, be sure not to take out；
5) Quality Control is evaporated：8 μ L TE/ water blank connect, and tube bottom at least 5 μ L of residue are completed in connection.
6) full-automatic nucleic acids instrument setting：Optimization injection voltage and time, PCR product can be on after diluteds Sample so that signal is in optimized analysis range.
2nd, the specificity of kit
1 material
Used in the process of experiment to virus with bacterium be shown in Table 4, these bacteriums or virus are relatively common, can be passed through The modes such as purchased in market obtain.
The used virus of 4 experiments of table and bacterium
2 methods
2.1 respectively with pig breeding and respiratory syndrome virus, swine fever virus, Nipah virus, African swine fever virus, aerogenesis The probe of six kinds of cause of diseases of capsular clostridium and actinobacillus pleuropneumoniae is to common swine disease cause of disease such as 2 porcine circovirus b types, pig Circovirus 2 d types, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, pig japanese encephalitis virus, porcine pseudorabies virus, Pig parvoviral carries out MLPA detections, verifies the specificity of probe.
2.2 probes mixed with six kinds of cause of diseases carry out MLPA detections to all viruses in table 4 and bacterium respectively.
3 results
3.1 are detected with any one group of probe of design, can only be amplified from corresponding viral template correspondingly sized Band, and to 2 porcine circovirus b types, 2 porcine circovirus d types, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, Pig japanese encephalitis virus, porcine pseudorabies virus, pig parvoviral are without amplified signal.Illustrate that probe specificity is good.
3.2 probes mixed with six kinds of cause of diseases carry out MLPA detections, Zhi Nengcong to all viruses in table 4 and bacterium respectively Band of corresponding size is amplified in corresponding viral template, and it is popular to 2 porcine circovirus b types, 2 porcine circovirus d types, pig Property diarrhea virus, transmissible gastro-enteritis virus, pig japanese encephalitis virus, porcine pseudorabies virus are without amplified signal.Show institute The method specificity of foundation is good.
3rd, the sensitivity of kit
1 material
Pig is bred with respiratory syndrome virus and swine fever virus by the preservation of this laboratory, clostridium perfringens toxoid A types (CVCC1126) it is provided with porcine contagious pleuropneumonia bacillus (CVCC3559) by Chinese veterinary medicament inspection.
2 methods
The structure of 2.1 plasmids and in-vitro transcription RNA
Pig breeds the preparation with respiratory syndrome virus M gene in-vitro transcription RNA：RT-PCR amplification pig breedings and breathing Viral (JX-7 plants) the M genes of trace integration sign simultaneously recycle amplified production, and length 339bp is cloned into pGEM-5zf carriers and (is purchased from Promega companies), positive recombinant plasmid is obtained after sequencing confirms, is named as pGEM-PRRSV-M.Using the plasmid of purifying as mould After plasmid is linearized with MluI enzymes, in-vitro transcription is carried out with the MEGAscript T7 kits of Ambion companies for plate, will In-vitro transcription product is precipitated with LiCl, and the ddH of no RNase is dissolved in after the washing of 70% ethyl alcohol2In O, nucleic acid electrophoresis detection RNA synthesis Integrality and correctness, and measure RNA concentration.Pig breeding and the RNA of respiratory syndrome virus M gene in-vitro transcription are ordered Entitled PRRSV-M-RNA.Pig breeds and SEQ ID NO in JX-7 plants of M gene orders of respiratory syndrome virus such as sequence table：24 It is shown.
The preparation of E 2 gene of Classical Swine Fever in-vitro transcription RNA：RT-PCR expands swine fever virus (SM plants) raq gene and recycles Amplified production, length 345bp are cloned into pGEM-5zf carriers (purchased from Promega companies), are obtained after sequencing confirms positive Recombinant plasmid is named as pGEM-CSFV-E2.Using the plasmid of purifying as template, after plasmid is linearized with MluI enzymes, use The MEGAscript T7 kits of Ambion companies carry out in-vitro transcription, in-vitro transcription product are precipitated with LiCl, 70% ethyl alcohol The ddH of no RNase is dissolved in after washing2In O, the integrality and correctness of nucleic acid electrophoresis detection RNA synthesis, and measure RNA concentration. Pig breeding and the RNA of respiratory syndrome virus M gene in-vitro transcription are named as CSFV-E2-RNA.E 2 gene of Classical Swine Fever SEQ ID NO in sequence such as sequence table：Shown in 25.
The preparation of pig Nipah virus N gene in-vitro transcriptions RNA：Since the country is there has been no Nipah virus, by database Sequence alignment, the sequence for choosing 292bp relatively conservative in Nipah virus N genes carries out outer-gene synthesis, is cloned into PGEM-5zf carriers (are purchased from Promega companies), are named as pGEM-NiPV-N.Using the plasmid of purifying as template, plasmid is used After the linearisation of MluI enzymes, in-vitro transcription is carried out with the MEGAscript T7 kits of Ambion companies, by in-vitro transcription product It is precipitated with LiCl, the ddH of no RNase is dissolved in after the washing of 70% ethyl alcohol2In O, the integrality and just of nucleic acid electrophoresis detection RNA synthesis True property, and measure RNA concentration.The RNA of pig Nipah virus N gene in-vitro transcriptions is named as NiPV-N-RNA.Pig Nipah virus N SEQ ID NO in gene order such as sequence table：Shown in 26.
The preparation of African swine fever virus p27 gene masculine recombinant plasmids：Due to the country, there has been no African swine fever viruses, pass through Sequence alignment in database, the sequence for choosing 278bp relatively conservative in African swine fever virus gene p27 carry out outer-gene Synthesis is cloned into pGEM-5zf carriers (purchased from Promega companies), is named as pGEM-ASFV-p27.African swine fever virus p27 SEQ ID NO in gene order such as sequence table：Shown in 27.
The preparation of C.perfringens plc gene masculine recombinant plasmids：PCR amplification C.perfringens (CVCC1126) Plc genes simultaneously recycle amplified production, length 211bp, pGEM-5zf carriers (purchased from Promega companies) are cloned into, through sequencing Positive recombinant plasmid is obtained after confirmation, is named as pGEM-C.perfringens-plc.C.perfringens pcl gene orders are such as SEQ ID NO in sequence table：Shown in 28.
The preparation of actinobacillus pleuropneumoniae ApxIVA gene masculine recombinant plasmids：PCR amplification pig pleuropneumonia unwrapping wire Bacillus (CVCC3559) ApxIVA genes simultaneously recycle amplified production, and length 223bp is cloned into pGEM-5zf carriers and (is purchased from Promega companies), positive recombinant plasmid is obtained after sequencing confirms, is named as pGEM-A.pleuropneumoniae- ApxIVA.SEQ ID NO in actinobacillus pleuropneumoniae ApxIVA gene orders such as sequence table：Shown in 29.
2.2 sensitivity are verified
With TE buffer solutions by the RNA of in-vitro transcription or recombinant plasmid dna from 100To 1010Doubling dilution is carried out, is then carried out MLPA reacts.
3 results
The minimum pig breeding that can detect 1 copy of this method and the swine fever virus of respiratory syndrome viral RNA, 1 copy RNA, the pig Nipah virus RNA of 1 copy, the African swine fever virus DNA of 1 copy, 74 copy C.perfringens DNA and 1 copy Actinobacillus pleuropneumoniae DNA.
Sequence table
<110>Zhejiang A ＆ F University
<120>The multiple linking probe amplification identification reagent box of a variety of pig Sudden Death Syndrome cause of diseases can be detected
<160> 29
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 1
cggatgaaag cccgcggcac t 21
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 2
catctgatgc atgcaccttg ac 22
<210> 3
<211> 26
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 3
cccatagacc tgtcaatagt agtagc 26
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 4
atggataccg agggaatagc 20
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 5
cttaccgatg aaaatgatac 20
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 6
agagaacatg catgagcttc a 21
<210> 7
<211> 24
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 7
tcctctttgc cattcatatc tagc 24
<210> 8
<211> 20
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 8
gtgcgggtaa tgatacggtt 20
<210> 9
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
cggctaaacc aaagttcgga t 21
<210> 10
<211> 45
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 10
gggttcccta agggttggag aaacctggaa attcatcacc tccag 45
<210> 11
<211> 49
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 11
atgccgtttg tgcttgctag gccgcatcta gattggatct tgctggcac 49
<210> 12
<211> 47
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 12
gggttcccta agggttggag gtaagtgcat tttggcaaat gagacag 47
<210> 13
<211> 59
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 13
gttacagaat agtagattca acggactgta acagagtcta gattggatct tgctggcac 59
<210> 14
<211> 52
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 14
gggttcccta agggttggag cttgatgcta ctctacagag aaattggccc aa 52
<210> 15
<211> 64
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 15
gagcccctta tatggtgctt cttgaagaat caattcagac ttctagattg gatcttgctg 60
gcac 64
<210> 16
<211> 61
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 16
gggttcccta agggttggat taatccagag cgcaagaggg ggctgatagt atttaggggt 60
t 61
<210> 17
<211> 65
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 17
tgaggtccat tacagctgta atgaacatta cgtcttatgt cctctagatt ggatcttgct 60
ggcac 65
<210> 18
<211> 66
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 18
gggttcccta agggttggat ttctgggatc ctgatacaga taataatttc tcaaaggata 60
atagtt 66
<210> 19
<211> 69
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 19
ggtatttagc ttattctata cctgacacag gggaatcaca aataagtcta gattggatct 60
tgctggcac 69
<210> 20
<211> 71
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 20
gggttcccta agggttggag aactttggtt tagccgagaa aataacgatt tgattattaa 60
atcattatta a 71
<210> 21
<211> 73
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 21
gtgaggataa agtcacggtt caaaattggt attcacacca agatcataaa tctagattgg 60
atcttgctgg cac 73
<210> 22
<211> 21
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 22
cggatgaaag cccgcggcac t 21
<210> 23
<211> 22
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 23
catctgatgc atgcaccttg ac 22
<210> 24
<211> 339
<212> DNA
<213>Pig breeds viral (PRRSV) with respiratory syndrome
<400> 24
aaggtgcttt tggcgttttc cattacctac acgccagtga tgatatatgc tctaaaggta 60
agtcgcggcc gactgctagg gcttctgcac cttttgatct ttctgaattg tgcttttacc 120
ttcgggtaca tgacattcgc gcactttgag agcacaaata gggtcgcgct cactatggga 180
gcagtagttg cacttctttg gggggtgtac tcagccatag aaacctggaa attcatcacc 240
tccagatgcc gtttgtgctt gctaggccgc aagtacatcc tggcccctgc ccaccacgtc 300
gaaagtgccg cgggctttca tccgattgcg gcaaatgat 339
<210> 25
<211> 345
<212> DNA
<213>Swine fever virus (CSFV)
<400> 25
agccctttcc gcacagaatg gattgtgtga ccaccacagt ggaaaatgaa gatttattct 60
attgtaagtt ggggggcaac tggacatgtg tgaaaggcga gccagtggtc tacacagggg 120
ggctagtaaa acaatgtaga tggtgtggct tcgacttcga tgggcctgac ggactcccgc 180
attaccccat aggtaagtgc attttggcaa atgagacagg ttacagaata gtagattcaa 240
cggactgtaa cagagatggc gttgtaatca gcacagaggg gagtcatgag tgcttgatcg 300
gtaacacgac tgtcaaggtg catgcatcag atgaaagatt gggcc 345
<210> 26
<211> 292
<212> DNA
<213>Pig Nipah virus (NiPV)
<400> 26
tagaaataat ctcagacatc ggaaactatg tcgaggaaac tggtatggca ggattcttcg 60
caaccatcag attcgggttg gagacaaggt atccagcact tgcactcaac gaattccaga 120
gtgacctcaa caccatcaaa agcttgatgc tactctacag agaaattggc ccaagagccc 180
cttatatggt gcttcttgaa gaatcaattc agactaaatt tgcccctgga ggttacccat 240
tattgtggag ctttgccatg ggtgtggcta ctactattga caggtctatg gg 292
<210> 27
<211> 278
<212> DNA
<213>African swine fever virus (ASFV)
<400> 27
atggataccg agggaatagc aaggttcacg ttctcgttaa accaaaagcg cagcttaatc 60
cagagcgcaa gagggggctg atagtattta ggggtttgag gtccattaca gctgtaatga 120
acattacgtc ttatgtccag atacgttgcg tccgtaatag gagtaatatc ttgtttacct 180
gctgtttgga tattgtgaga gttctcggga aaatgttgtg aaaggaattt cgggttggta 240
tggctgcacg ttcgctgcgt atcattttca tcggtaag 278
<210> 28
<211> 211
<212> DNA
<213>C.perfringens (Clostridium perfringens)
<400> 28
agagaacatg catgagcttc aattaggttc tacttatcca gattatgata agaatgcata 60
tgatctatat caagatcatt tctgggatcc tgatacagat aataatttct caaaggataa 120
tagttggtat ttagcttatt ctatacctga cacaggggaa tcacaaataa gaaaattttc 180
agcattagct agatatgaat ggcaaagagg a 211
<210> 29
<211> 223
<212> DNA
<213>Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae)
<400> 29
gtgcgggtaa tgatacggtt aatggcggta atggcgatga caccctcatc ggcggcaaag 60
gtaatgatat tctaagaggt ggctacggtg cggacaccta tatctttagc aaaggacacg 120
gacaggatat cgtttatgaa gataccaata atgataaccg cgcaagagat atcgacacct 180
taaaatttac tgatattaat ttatccgaac tttggtttag ccg 223

Claims (4)

  1. A kind of 1. multiple linking probe amplification identification reagent box that can detect a variety of pig Sudden Death Syndrome cause of diseases, which is characterized in that the examination Agent box includes：
    (1) reverse transcription and pre- amplification primer mixed liquor；The mixed liquor includes sequence SEQ ID NO：Pig is used for shown in 1~9 Breeding and respiratory syndrome virus, swine fever virus, the reverse transcription primer of Nipah virus and African swine fever virus, perfringens shuttle Bacterium and the pre- amplimer of actinobacillus pleuropneumoniae；
    (2) probe mixed liquor；The mixed liquor includes sequence SEQ ID NO：Being bred for detecting pig with exhaling shown in 10~21 Inhale trace integration sign virus, swine fever virus, Nipah virus, African swine fever virus, C.perfringens and pig pleuropneumonia unwrapping wire bar The left side of bacterium and right side probe；
    (3) MLPA buffer solutions；
    (4) buffer solution A is connected；
    (5) buffer solution B is connected；
    (6) ligase Ligase-65；
    (7) PCR reaction mixtures, the mixed liquor include sequence SEQ ID NO:Universal primer shown in 22 and 23；
    (8) SALSA polymerases；
    (9) negative control；
    (10) positive control.
  2. 2. kit according to claim 1, which is characterized in that sequence SEQ ID NO：1 is that pig breeding is comprehensive with respiratory tract Simulator sickness virus reverse transcriptase primer；Sequence SEQ ID NO：2 be swine fever virus reverse transcription primer；Sequence SEQ ID NO：3 be Ni Pa Virus reverse transcriptase primer；Sequence SEQ ID NO：4 and SEQ ID NO：5 be the forward direction that African swine fever virus expands in advance respectively and anti- To primer；Sequence SEQ ID NO：6 and SEQ ID NO：7 be the forward and reverse primer that C.perfringens expands in advance respectively； Sequence SEQ ID NO：8 and SEQ ID NO：9 be the forward and reverse primer that actinobacillus pleuropneumoniae expands in advance respectively；Sequence Arrange SEQ ID NO：10 and SEQ ID NO：11 be respectively left side probe and the right side of the detection pig breeding with respiratory syndrome virus Side probe；Sequence SEQ ID NO：12 and SEQ ID NO：13 be respectively the detection left side probe of swine fever virus and right side probe； Sequence SEQ ID NO：14 and SEQ ID NO：15 be respectively the detection left side probe of Nipah virus and right side probe；Sequence SEQ ID NO：16 and SEQ ID NO：17 be respectively the detection left side probe of African swine fever virus and right side probe；Sequence SEQ ID NO：18 and SEQ ID NO：19 be respectively the detection left side probe of C.perfringens and right side probe；Sequence SEQ ID NO： 20 and SEQ ID NO：21 be respectively the detection left side probe of actinobacillus pleuropneumoniae and right side probe；Sequence SEQ ID NO：22 and SEQ ID NO：23 be general forward and reverse primer respectively；Wherein, sequence SEQ ID NO：11, SEQ ID NO： 13, SEQ ID NO：15, SEQ ID NO：17, SEQ ID NO：19 and SEQ ID NO：21 5 ' ends carry out phosphatizing treatment.
  3. 3. kit according to claim 1, which is characterized in that the positive control is gene in-vitro transcription RNA and sun The mixture of property recombinant plasmid dna；Wherein gene in-vitro transcription RNA refer to pig breeding and the M genes of respiratory syndrome virus, The raq gene of swine fever virus and N genes the in-vitro transcription RNA, positive recombinant plasmid DNA of Nipah virus refer to African swine fever virus P27 genes, C.perfringens plc genes and actinobacillus pleuropneumoniae ApxIVA genes positive recombinant plasmid DNA。
  4. 4. detect the multiple linking probe augmentation detection side of a variety of pig Sudden Death Syndrome cause of diseases simultaneously using kit described in claim 1 Method, which is characterized in that its operating process is as described below：
    (1) paramagnetic particle method extraction sample DNA/RNA；
    Extracts kit is total to the magnetic bead DNA/RNA of TIANGEN companies, using the grand NP968 Full automatic instrument for extracting nucleic acid in day simultaneously The DNA and RNA in sample are extracted, obtains 200 μ L samples；
    (2) RNA reverse transcriptions are expanded into cDNA and in advance
    One-step method reverse transcription RT-PCR reactions are carried out according to QIAGEN OneStep Ahead RT-PCR Kit specifications；It prepares 25 μ L reaction systems：10 μ L OneStep Ahead RT-PCR Master Mix, 1 μ L OneStep Ahead RT-Mix, 5 μ L DNA or RNA, 5 μ L reverse transcriptions and pre- amplification primer mixed liquor (final concentration of 0.5 μM of every primer), 4 μ L H2O is supplied；Instead Answer condition：50 DEG C of 10min, 95 DEG C of 5min；95 DEG C of 15s, 55 DEG C of 20s, 72 DEG C of 20s, 40 cycles；72℃2min；
    (3) MLPA is detected
    A, DNA is denaturalized
    0.2mL PCR reaction tubes are taken, often pipe adds in 0.5 μ L DNA solutions and 4.5 μ L TE, 98 DEG C of denaturation 5min are down to room temperature 25 ℃；
    B, probe and sample DNA hybridize
    Prepare the probe mixed liquor of 3 μ L mixings：+ 1.5 μ L probe mixed liquors of 1.5 μ L MLPA buffer solutions；Probe mixture is added in In above-mentioned PCR pipe, 95 DEG C of incubation 1min, 60 DEG C of hybridization 1-16h, 54 DEG C incubate；
    C, the connection of hybridization probe
    Prepare 32 μ L connection enzymatic mixtures：25μL dH2+ 1 μ L ligases of+3 μ L connections buffer solution B of O+3 μ L connections buffer solution A Ligase-65；PCR instrument temperature is down to 54 DEG C, opens pipe lid, adds in 32 μ L connection enzymatic mixtures, and 54 DEG C incubate 15min, 98 DEG C Heat 5min inactivation ligases, 20 DEG C of incubations；
    D, the PCR amplification of linking probe
    Prepare 10 μ L PCR mixed liquors：7.5μL dH2+ 0.5 μ L SALSA polymerases of O+2 μ L PCR reaction mixtures；Take out PCR Pipe, adds in 10 μ L PCR mixtures at room temperature；Start PCR reactions, reaction condition is：95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 60s, 35 A cycle；72 DEG C of incubation 20min, are down to 15 DEG C；
    E, full-automatic nucleic acids instrument analysis：
    Pcr amplification product is analyzed with full-automatic nucleic acids instrument (Qsep100DNA Analyzer)；
    (4) result description and judgement
    A, quality control standard：
    Positive control has specific amplification band at 94bp, 106bp, 116bp, 126bp, 135bp, 144bp；
    Negative control is without specific amplification band；
    If negative control and positive condition are unsatisfactory for conditions above, this time experiment is considered as invalid；
    B, result judges：
    It is positive：There is specific amplification band at 94bp, represent that there are pig breedings and respiratory syndrome virus in sample； There is specific amplification band at 106bp, represent that there are swine fever virus in sample；There is specific amplification band at 116bp, represent There are Nipah virus in sample；There is specific amplification band at 126bp, represent that there are African swine fever viruses in sample； There is specific amplification band at 135bp, represent that there are C.perfringens in sample；There is specific amplification band at 144bp, Represent that there are actinobacillus pleuropneumoniaes in sample；MLPA amplified productions can be sequenced, further confirmed that；
    It is negative：Without specific amplification band, show in sample without pig breeding and respiratory syndrome virus, swine fever virus, Ni Pa Virus, African swine fever virus, C.perfringens and actinobacillus pleuropneumoniae.
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CN109207635A (en) * 2018-09-19 2019-01-15 浙江农林大学 Detect the MLPA detection kit of pig virus breeding difficulty syndrome etiology nucleic acid
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CN108913563A (en) * 2018-07-05 2018-11-30 宁夏大学 A kind of micro-fluidic genetic chip of dish-style and related kit and purposes
CN109161613A (en) * 2018-09-19 2019-01-08 浙江农林大学 A kind of MLPA detection kit detecting pig virus diarrhoea syndrome etiology nucleic acid
CN109207635A (en) * 2018-09-19 2019-01-15 浙江农林大学 Detect the MLPA detection kit of pig virus breeding difficulty syndrome etiology nucleic acid
CN109207635B (en) * 2018-09-19 2021-10-22 浙江农林大学 MLPA detection kit for detecting pathogenic nucleic acid of porcine viral reproductive disorder syndrome
CN109161613B (en) * 2018-09-19 2021-10-22 浙江农林大学 MLPA detection kit for detecting pathogenic nucleic acid of porcine viral diarrhea syndrome
CN109706269A (en) * 2019-02-06 2019-05-03 浙江农林大学 The multiple linking probe that a variety of fowl respiratory pathogens can be detected expands identification reagent box
CN109706269B (en) * 2019-02-06 2022-07-01 浙江农林大学 Multiplex connection probe amplification identification kit capable of detecting various avian respiratory pathogens
CN110302371A (en) * 2019-08-21 2019-10-08 军事科学院军事医学研究院军事兽医研究所 Inactivate purposes of the ASFV as the Immunization protective ingredient of combination vaccine
CN110302371B (en) * 2019-08-21 2023-05-26 军事科学院军事医学研究院军事兽医研究所 Use of inactivated ASFV as immune toxin-counteracting protective component of composite vaccine
CN114686608A (en) * 2020-12-30 2022-07-01 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） Rapid visual detection method for actinobacillus pleuropneumoniae based on CRISPR-Cas12a
CN115976281A (en) * 2022-11-18 2023-04-18 中国农业大学 Primer group for realizing simultaneous detection of 15 porcine pathogens through high-throughput targeted sequencing and application
CN115976281B (en) * 2022-11-18 2023-07-25 中国农业大学 Primer group for simultaneously detecting 15 pathogens of pigs through high-throughput targeted sequencing and application

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