CN109161613B - MLPA detection kit for detecting pathogenic nucleic acid of porcine viral diarrhea syndrome - Google Patents

Abstract

The invention relates to the field of inspection and quarantine, and aims to provide an MLPA (multi-layered double-effect amplification) detection kit for detecting pathogenic nucleic acid of porcine viral diarrhea syndrome. The kit comprises: (1) reverse transcription and pre-amplification mixture: the sequences of the reverse transcription and preamplification primers are shown as SEQ ID NO: 1-12; wherein, the reverse transcription primer is a random primer with 12 basic groups; (2) multiplex ligation probe mixture: wherein, the sequence of the probe is shown as SEQ ID NO: 13-24, and the sequence of the universal primer is shown as SEQ ID NO: 25-26; (3) MLPA buffer solution; (4) connecting buffer solutions A and B; (5) ligase Ligase-65; (6) a PCR reaction mixed solution which comprises a universal primer shown in a sequence table SEQ ID NO. 25-26; (7) SALSA polymerase; (8) negative control; (9) a positive control comprising a positive control plasmid of 6 pathogens of porcine viral diarrhea syndrome. Compared with the existing method, the invention has the advantages of high flux, cost saving, good specificity and high sensitivity.

Description

MLPA detection kit for detecting pathogenic nucleic acid of porcine viral diarrhea syndrome

Technical Field

The invention provides a multiple connection probe amplification identification detection kit for simultaneously detecting Porcine Epidemic Diarrhea Virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine Rotavirus (RV), porcine delta coronavirus (PDCoV), porcine bocavirus (PBoV) and porcine norovirus (NoV)6, can realize simultaneous detection of 6 pathogens causing porcine viral diarrhea syndrome, and belongs to the field of inspection and quarantine.

Background

The porcine viral diarrhea is the most important disease harming the pig industry in China at present, the porcine viral diarrhea can harm pigs at any age, particularly suckling and weaning piglets which frequently occur from 12 months to 1-2 months next year, the morbidity and mortality of the porcine viral diarrhea are high, the incidence and the propagation are rapid and difficult to control, the porcine viral diarrhea greatly harms the safety of pig groups in large-scale farms, and the great loss is brought to the pig industry [1 ]. Once infected with the disease, piglets often show watery diarrhea, dehydrate until death, have very high morbidity and mortality rate, and often bring devastating attacks to the farms. Common etiologies of porcine viral diarrhea syndrome are: porcine Epidemic Diarrhea Virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine Rotavirus (RV), porcine delta coronavirus (PDCoV), porcine bocavirus (PBoV), porcine norovirus (NoV), and the like. Because the syndrome is quick in onset, the pathogenic cause needs to be determined in a short time and the disease is prevented and treated, and no method can be used for carrying out differential diagnosis on various disease pathogens causing the porcine viral diarrhea at one time.

Therefore, a detection method capable of accurately, quickly and efficiently distinguishing different pathogens is established, and is very important for preventing and generating porcine viral diarrhea.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects in the prior art and provides an MLPA (multiple-layer linear amplification) detection kit for detecting pathogenic nucleic acid of porcine viral diarrhea syndrome. The kit provides a detection means for simultaneously detecting Porcine Epidemic Diarrhea Virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine Rotavirus (RV), porcine delta coronavirus (PDCoV), porcine bocavirus (PBoV) and porcine norovirus (NoV) for clinic.

In order to solve the technical problems, the invention adopts the following technical scheme:

provides an MLPA detection kit for detecting pathogenic nucleic acid of porcine viral diarrhea syndrome, which comprises:

(1) reverse transcription and pre-amplification mixture:

the mixture contains reverse transcription and pre-amplification primers, the sequences of which are shown as SEQ ID NO: 1-12 (see table 1), respectively used for reverse transcription and pre-amplification; wherein, the reverse transcription primer is a random primer with 12 basic groups;

(2) multiplex ligation probe mixture:

the mixed solution comprises a probe and a universal primer; wherein, the sequence of the probe is shown as SEQ ID NO: 13-24 (see table 2), and the sequence of the universal primer is shown as SEQ ID NO:25 to 26 (see Table 3);

(3) MLPA buffer solution;

(4) connecting buffer solutions A and B;

(5) ligase Ligase-65;

(6) a PCR reaction mixed solution which comprises a universal primer shown in a sequence table SEQ ID NO. 25-26;

(7) SALSA polymerase;

(8) negative control;

(9) positive controls comprised of Porcine Epidemic Diarrhea Virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine Rotavirus (RV), porcine delta coronavirus (PDCoV), porcine bocavirus (PBocV), and porcine Norovirus (NV) positive control plasmids.

In the invention, the sequence of SEQ ID NO: 1-2 is a porcine bocavirus pre-amplification primer; the sequence of SEQ ID NO: 3-4 are porcine norovirus pre-amplification primers; the sequence of SEQ ID NO: 5-6 are porcine delta coronavirus pre-amplification primers; the sequence of SEQ ID NO: 7-8 are porcine epidemic diarrhea virus pre-amplification primers respectively; the sequence of SEQ ID NO: 9-10 is a porcine rotavirus pre-amplification primer; the sequence of SEQ ID NO: 11-12 are porcine transmissible gastroenteritis virus pre-amplification primers; the sequence of SEQ ID NO: 13-14 are a left probe and a right probe for detecting the porcine bocavirus; the sequence of SEQ ID NO: 15-16 are left and right probes for detecting the porcine norovirus respectively; the sequence of SEQ ID NO: 17-18 are a left side probe and a right side probe for detecting the porcine delta coronavirus respectively; the sequence of SEQ ID NO: 19-20 are respectively a left side probe and a right side probe for detecting the porcine epidemic diarrhea virus; the sequence of SEQ ID NO: 21-22 are respectively a left side probe and a right side probe for detecting the porcine rotavirus; the sequence of SEQ ID NO: 23-24 are respectively a left side probe and a right side probe for detecting the transmissible gastroenteritis virus; wherein, the SEQ ID NO: 14. 16, 18, 20, 22 and 24' ends are subjected to phosphorylation treatment; the sequence of SEQ ID NO: 25. 26 are the forward and reverse primers, respectively, for universal PCR amplification.

In the invention, the positive control in the kit is a recombinant plasmid mixture obtained by connecting a specific gene segment of virus nucleic acid RNA after reverse transcription with plasmid; the specific genes are S gene of Porcine Epidemic Diarrhea Virus (PEDV), N gene of porcine transmissible gastroenteritis virus (TGEV), NSP1 gene of porcine Rotavirus (RV), N gene of porcine delta coronavirus (PDCoV), NS1 gene of porcine bocavirus (PBoV) and RdRp gene of porcine Norovirus (NV).

The invention further provides an MLPA detection test method for simultaneously detecting 6 porcine viral diarrhea syndrome pathogenic nucleic acids by using the kit, which comprises the following steps:

(1) extracting DNA and RNA of a sample by a magnetic bead method;

simultaneously extracting DNA and RNA in a sample by using a magnetic bead DNA and RNA co-extraction kit and a full-automatic nucleic acid extractor to obtain a 200 mu L sample;

(2) reverse transcription of RNA into cDNA and preamplification

Carrying out one-step reverse transcription RT-PCR reaction;

prepare 25 μ L reaction system: mu.L of OneStep Ahead RT-PCR Master Mix, 1. mu.L of OnStep Ahead RT-Mix, 5. mu.L of DNA or RNA, 1. mu.L of random reverse transcription primer and 5. mu.L of pre-amplification primer mixture (final concentration: 0.5. mu.M per primer), 3. mu. L H₂O is complemented;

reaction conditions are as follows: 10min at 50 ℃ and 5min at 95 ℃; at 95 ℃ for 15s, at 55 ℃ for 20s, at 72 ℃ for 20s, for 40 cycles; 2min at 72 ℃;

(3) MLPA amplification and detection

a) Denaturation of DNA

Taking 0.2mL PCR reaction tubes, adding 0.5 mu L DNA solution and 4.5 mu L TE into each tube, denaturing at 98 ℃ for 5min, and cooling to room temperature of 25 ℃;

b) hybridization of the Probe with sample DNA

Preparing 3 mu L of mixed probe liquid: 1.5. mu.L MLPA buffer solution + 1.5. mu.L probe mixture;

adding the probe mixture into the PCR reaction tube, incubating at 95 ℃ for 1min, hybridizing at 60 ℃ for 1-16h, and incubating at 54 ℃;

c) ligation of hybridization probes

Prepare 32 μ L ligase mix: 25 μ L dH₂O + 3. mu.L of a ligation buffer A + 3. mu.L of a ligation buffer B +1. mu.L of Ligase Ligase-65;

cooling the temperature of the PCR instrument to 54 ℃, opening a tube cover, adding 32 mu L of ligase mixture, incubating at 54 ℃ for 15min, heating at 98 ℃ for 5min to inactivate ligase, and incubating at 20 ℃;

d) PCR amplification of ligated probes

Prepare 10 μ L of PCR mix: 7.5 μ L dH₂O + 2. mu.L of PCR reaction mixture + 0.5. mu.L of SALSA polymerase;

taking out the PCR tube, and adding 10 mu L of PCR mixture at room temperature; the PCR reaction was started under the following conditions: 30s at 95 ℃, 30s at 60 ℃, 60s at 72 ℃ and 35 cycles; incubating at 72 deg.C for 20min, and cooling to 15 deg.C;

e) analysis by a full-automatic nucleic acid analyzer:

taking 20 mu L of PCR amplification product, and analyzing by using a full-automatic nucleic acid analyzer;

(4) result description and determination

a) Quality control standard:

the positive control has specific amplification bands at positions of 102bp, 110bp, 117bp, 124bp, 131bp and 138 bp;

negative control no specific amplification band;

if the negative control and the positive condition do not satisfy the above conditions, the test is regarded as invalid;

b) and (5) judging a result:

positive: a specific amplification band exists at a position of 102bp, which indicates that the porcine bocavirus exists in a sample; a specific amplification band exists at a position of 110bp, which indicates that the porcine norovirus exists in the sample; a specific amplification band exists at 117bp, which indicates that the porcine delta coronavirus exists in the sample; a specific amplification band exists at 124bp, which indicates that porcine epidemic diarrhea virus exists in the sample; a specific amplification strip is arranged at 131bp, which indicates that the porcine rotavirus exists in the sample; a specific amplification band exists at 138bp, which indicates that the transmissible gastroenteritis virus exists in the sample; meanwhile, MLPA amplification products can be sequenced and further confirmed;

negative: and (3) no specific amplification strip indicates that the sample does not contain porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus, porcine delta coronavirus, porcine bocavirus and porcine norovirus.

Compared with the prior art, the method has the following advantages and effects:

1) high flux. One reaction can detect 6 pathogens simultaneously, 96 samples can be detected simultaneously each time by using a full-automatic nucleic acid analyzer, and a high-throughput detection technology is provided for differential diagnosis and emergency diagnosis of the pathogens.

2) The cost is saved. Generally, one sample is used for detecting 6 disease pathogenic nucleic acids by a fluorescence quantitative method, the cost is usually 200X 6-1200 yuan, and the cost of the kit is about 400 yuan due to the reaction in one tube.

3) The specificity is good. Through sequence comparison and blast analysis, the design of the probe is enhanced, and the probe is ensured to be only combined with the target gene.

4) The sensitivity is high. The conventional MLPA method requires at least 6000 copies of the target DNA. The invention enriches the target gene by adding the step of RT-PCR, and about 10 copies of the target gene can be detected at the minimum.

Drawings

FIG. 1 shows the PCR validation of the positive control recombinant plasmid construction for 6 diseases;

in the figure, M is DL2000 DNA Marker; 1 is PDCoV-N, 2 is TGEV-N, 3 is RV-NSP1, 4 is PEDV, 5 is PBoV, and 6 is NoV.

FIGS. 2.1 to 2.6 are peak diagrams and simulated electrophoretograms (single template) for specificity verification of the porcine viral diarrhea syndrome MLPA method;

the figure shows the results of detection according to MLPA reaction program by adding constructed TGEV, RV, PEDV, PDCoV, NoV and PBoV gene positive plasmids into a system with 6 pairs of preamplification primers and 6 pairs of probes respectively.

FIG. 3 peak and simulated electropherograms (mixed templates) for specificity validation of the porcine viral diarrhea syndrome MLPA method.

The constructed TGEV-N, RV-Nsp1, PEDV-S, PDCoV-N, NoV-RdRp and PBoV-NS1 gene positive plasmids are added into a system with 6 pairs of pre-amplification primers and 6 pairs of probes together, and the detection result is carried out according to an MLPA reaction program. The porcine bocavirus has a specific amplification band at 102 bp; the porcine norovirus has a specific amplification band at 110 bp; the pig delta coronavirus has a specific amplification band at 117 bp; the porcine epidemic diarrhea virus has a specific amplification band at 124 bp; the porcine rotavirus has a specific amplification band at 131 bp; the porcine transmissible gastroenteritis virus has a specific amplification band at 138 bp.

Detailed Description

The multiplex ligation-dependent probe amplification (MLPA) is a new technology for qualitative and quantitative analysis of target sequences in nucleic acids to be detected with high throughput. The technology combines the hybridization detection of nucleic acid and PCR chain amplification, thereby realizing the high-efficiency specificity analysis of target molecules, and detecting and quantitatively analyzing 45 different target genes in the same reaction tube. The basic principle of MLPA includes hybridization between probe and target sequence DNA, connection, PCR amplification, separation of product by capillary electrophoresis and data collection, and analysis of the collected data by DNA analysis software. Each MLPA probe comprises two oligonucleotide fragments, one chemically synthesized and one derived from M13 phage; each probe comprises a primer sequence and a specific sequence. In the MLPA reaction, both oligonucleotide fragments hybridize to the target sequence, followed by ligation of the two part probes using ligase. The ligation reaction is highly specific, and only when the two probes are completely hybridized with the target sequence, namely the target sequence is completely complementary with the probe specific sequence, the ligase can connect the two probes into a complete nucleic acid single chain; on the other hand, if the target sequence is not completely complementary to the probe sequence, hybridization will be incomplete even if there is a single base difference, and the ligation reaction will not proceed. After the connection reaction is finished, the connected probes are amplified by using a pair of universal primers, and the length of an amplification product of each pair of probes is unique and ranges from 90 bp to 480 bp. Finally, the amplified products are separated by capillary electrophoresis and analyzed by software to obtain a conclusion.

The invention constructs a detection method for simultaneously detecting the 6 porcine viral diarrhea syndrome based on the MLPA technology, and provides a high-throughput diagnosis technology with strong specificity and high sensitivity for diseases with the symptoms.

In order to improve the detection sensitivity of MLPA, a pair of pre-amplification primers (the sequence is shown in table 1) is designed for each disease according to specific sequences on GenBank of 6 diseases through Blast analysis, the sequences comprise sequences of MLPA probes, the MLPA is synthesized and pre-amplified through cDNA based on a one-step method, then the MLPA is carried out according to the operation flow of the invention, the probes (the sequence is shown in table 2) are denatured, hybridized and connected, PCR amplification is carried out by using general primers (the sequence is shown in table 3), and then capillary electrophoresis detection is carried out.

TABLE 16 porcine viral diarrhea multiplex ligation Probe amplification technology (MLPA) Pre-amplification primer sequences (5 '-3')

TABLE 26 porcine viral diarrhea multiplex ligation Probe amplification technology (MLPA) Probe sequence (5 '-3')

Wherein, the SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: carrying out phosphorylation treatment on the 5' end of the 24;

TABLE 3 Universal primer sequences

The invention adopts a multiple connection probe amplification technology to establish a multiple connection probe amplification detection method for simultaneously detecting porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus, porcine delta coronavirus, porcine bocavirus and porcine norovirus 6, and assembles a kit, wherein the kit comprises the following components:

(1) a reverse transcription and pre-amplification primer mixed solution, which comprises pre-amplification primers of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus, porcine delta coronavirus, porcine bocavirus and porcine norovirus, wherein the primer sequences are shown in table 1, and the concentration of each primer in the mixed solution is 2.5 mu M;

(2) a probe mixture comprising left and right probes for detecting porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus, porcine delta coronavirus, porcine bocavirus and porcine norovirus, the sequences of said probes being shown in table 2, wherein the sequences of said probes are shown in SEQ ID NO: 14. 16, 18, 20, 22 and 24' ends are subjected to phosphorylation treatment; the concentration of each probe in the mixture was 1.33 nM;

(3) MLPA buffer solution;

(4) connecting a buffer solution A;

(5) connecting a buffer solution B;

(6) ligase Ligase-65;

(7) PCR reaction mixed liquor, which comprises universal primers shown in sequence tables SEQ ID NO 25 and 26;

(8) SALSA polymerase;

(9) negative control: TE;

(10) positive control: is a mixture of TGEV-N, RV-Nsp1, PEDV-S, PDCoV-N, NoV-RdRp and PBoV-NS1 gene positive plasmid DNA.

The following describes in detail the MLPA detection method for simultaneously detecting 6 porcine viral diarrhea syndrome pathogenic nucleic acids by using the kit of the present invention with reference to specific examples.

First, the kit's instructions

1 composition of the kit

Among them, MLPA buffer, ligation buffer A, ligation buffer B, Ligase Ligase-65 and SALSA polymerase were purchased from MRC-Holland, Inc.

Storage conditions were as follows:

except for the positive control, the other components were stored at-15 ℃ to-25 ℃. Positive controls were stored at-80 ℃.

In order to ensure the experimental effect, the kit is used within one year.

2 detection (Using) method

2.1 extracting DNA/RNA of a sample by a magnetic bead method;

DNA and RNA in the sample were simultaneously extracted using a magnetic bead DNA/RNA co-extraction kit (cat # DP422) from TIANGEN using a Tianlong NP968 full-automatic nucleic acid extractor, to give 200. mu.L of the sample.

2.2 reverse transcription of RNA into cDNA and Pre-amplification

According to QIAGENOneStep Ahead RT-PCR Kit instructions for one-step reverse transcription RT-PCR reaction. Prepare 25 μ L reaction system: mu.L of OneStep Ahead RT-PCR Master Mix, 1. mu.L of OnStep Ahead RT-Mix, 5. mu.L of DNA or RNA, 1. mu.L of random reverse transcription primer and 5. mu.L of pre-amplification primer mixture (final concentration: 0.5. mu.M per primer), 3. mu. L H₂And (4) complementing O. Reaction conditions are as follows: 10min at 50 ℃ and 5min at 95 ℃; at 95 ℃ for 15s, at 55 ℃ for 20s, at 72 ℃ for 20s, for 40 cycles; 72 ℃ for 2 min.

2.3MLPA amplification and detection

2.3.1DNA denaturation

0.2mL of PCR reaction tube was taken, 0.5. mu.L of DNA solution and 4.5. mu.L of TE were added to each tube, denatured at 98 ℃ for 5min, and cooled to room temperature of 25 ℃.

2.3.2 hybridization of Probe to sample DNA

Preparing 3 mu L of mixed probe liquid: mu.L MLPA buffer + 1.5. mu.L probe mix. The probe mixture was added to the PCR tube and incubated at 95 ℃ for 1min, 60 ℃ for hybridization for 1-16h and 54 ℃.

2.3.3 ligation of hybridization probes

Prepare 32 μ L ligase mix: 25 μ L dH₂O + 3. mu.L ligation buffer A + 3. mu.L ligation buffer B +1. mu.L Ligase Ligase-65. The temperature of the PCR instrument was lowered to 54 deg.C, the lid was opened, 32. mu.L of the ligase mixture was added, incubated at 54 deg.C for 15min, heated at 98 deg.C for 5min to inactivate the ligase, and incubated at 20 deg.C.

2.3.4 PCR amplification of ligation probes

Prepare 10 μ L of PCR mix: 7.5 μ L dH₂O + 2. mu.L of PCR reaction mix + 0.5. mu.L of SALSA polymerase. The PCR tube was removed and 10. mu.L of the PCR mixture was added at room temperature. The PCR reaction was started under the following conditions: 30s at 95 ℃, 30s at 60 ℃, 60s at 72 ℃ and 35 cycles; incubating at 72 deg.C for 20min, and cooling to 15 deg.C.

2.3.5 analysis by a full-automatic nucleic acid analyzer:

20. mu.L of the PCR amplification product was analyzed by a fully automatic nucleic acid Analyzer (Qsep100 DNA Analyzer).

2.4 results description and determination

2.4.1 quality control Standard:

the positive control has specific amplification bands at positions of 102bp, 110bp, 117bp, 124bp, 131bp and 138 bp.

Negative control no specific amplified band.

If the negative control and the positive condition do not satisfy the above conditions, the test is regarded as invalid.

2.4.2 judging the result:

positive: a specific amplification band exists at a position of 102bp, which indicates that the porcine bocavirus exists in a sample; there is a specific amplification band at 110bp, indicating the presence of the porcine norovirus in the sample. A specific amplification band exists at 117bp, which indicates that the porcine delta coronavirus exists in the sample; a specific amplification band exists at 124bp, which indicates that porcine epidemic diarrhea virus exists in the sample; a specific amplification strip is arranged at 131bp, which indicates that the porcine rotavirus exists in the sample; a specific amplification band at 138bp indicates that the transmissible gastroenteritis virus of swine exists in the sample. Meanwhile, MLPA amplification products can be sequenced and further confirmed.

Negative: and (3) no specific amplification strip indicates that the sample does not contain porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus, porcine delta coronavirus, porcine bocavirus and porcine norovirus.

Note that:

1) DNA sample:

a. the contents of salt ions, alcohols and the like in the DNA solution are reduced as much as possible.

b. TE is used for dissolving or diluting DNA instead of water, so that depurination of DNA at high temperature is avoided.

c. All DNA samples were diluted to approximately equal concentrations (20-40 ng/ul suggested) prior to the experiment.

2) The probe, the Ligase Ligase-65 and the connecting buffer solution A are packaged before use, so that repeated freeze thawing is avoided;

3) before the buffer solution and the reagent are used, the buffer solution and the reagent are vibrated, uniformly mixed and centrifuged, and the buffer solution and the reagent are lightly blown and uniformly mixed by a gun head during mixing, so that bubbles are prevented from being generated or the buffer solution and the reagent are beaten on the tube wall, and all enzyme-containing steps cannot be centrifuged;

4) when the ligase is added for reaction, the PCR tube is placed in a PCR instrument and is not taken out;

5) evaporation quality control: 8 μ L TE/water blank connections, leaving at least 5 μ L of the tube bottom after completion of the connection.

6) Setting a full-automatic nucleic acid analyzer: the injection voltage and time are optimized, and the PCR product can be diluted by a diluent and then loaded, so that the signal is in the optimal analysis range.

Second, specificity of the kit

1 Material

The viruses and bacteria used in this experiment are shown in Table 4.

TABLE 4 viruses and bacteria used in this experiment

Note: the viruses or strains described in Table 4 are all well known in the art and are available to those skilled in the art by commercial means. The applicant has committed to provide a sample of the above-mentioned viruses or strains to the public of society within 20 years from the date of filing of the present application.

2 method

2.1 detecting MLPA by six pathogeny single probes of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus, porcine delta coronavirus, porcine bocavirus and porcine norovirus respectively aiming at common porcine pathogeny such as porcine circovirus type 2b, porcine circovirus type 2d, porcine Japanese encephalitis virus, porcine pseudorabies virus, porcine parvovirus, porcine reproductive and respiratory syndrome virus, swine fever virus, clostridium perfringens and porcine actinobacillus pleuropneumoniae to verify the specificity of the probes.

2.2 MLPA detection of all viruses and bacteria in Table 4 with mixed probes of six pathogens, respectively.

3 results

3.1 any group of probes is used for detection, only strips with corresponding sizes can be amplified from corresponding virus templates, and no amplification signals are generated on porcine circovirus type 2b, porcine circovirus type 2d, porcine Japanese encephalitis virus, porcine pseudorabies virus, porcine parvovirus, porcine reproductive and respiratory syndrome virus, swine fever virus, clostridium perfringens and actinobacillus pleuropneumoniae. Indicating that the probe specificity was good.

3.2 the MLPA detection is carried out on all the viruses and bacteria in the table 4 by using probes mixed with six pathogens, only strips with corresponding sizes can be amplified from corresponding virus templates, and no amplification signals exist on porcine circovirus type 2b, porcine circovirus type 2d, porcine Japanese encephalitis virus, porcine pseudorabies virus, porcine parvovirus, porcine reproductive and respiratory syndrome virus, classical swine fever virus, clostridium perfringens and actinobacillus pleuropneumoniae. Indicating that the established method is good in specificity.

Third, the sensitivity of the kit

1 Material

See table 4.

2 method

2.1 construction of plasmids

The amplified TGEV-N, RV-Nsp1, PEDV-S, PDCoV-N, NoV-RdRp and PBoV-NS1 gene fragments are connected with pZERO-blunt vectors, transformed into Escherichia coli DH5a, recombinant plasmids are extracted, and PCR verification and sequencing verification are carried out.

2.2 sensitivity verification

Measuring the light absorption values A260 and A280 of the recombinant plasmid by using an ultraviolet spectrophotometer, calculating the copy number of the recombinant plasmid, and diluting the positive recombinant plasmid to 10 by using an EASY Dilution reagent¹⁰Copies. mu.L^－1Then, the gradient was diluted 10 times to 10⁸，10⁷，10⁶，10⁵，10⁴，10³，10²，10¹，10⁰Copies. mu.L^－1MLPA reactions were performed using 9 copy number gradients as standard templates. Plasmid mass concentration (g.L)^－1)＝λ(A260)×50(g·L^－1) The/1000 Xstandard template release dilution factor. Copy number calculation formula: copy number (. mu.L)^－1) Plasmid concentration (g. mu.L)^－1) X Afugardo constant/recombinant plasmid molecular weight.

3 results

The method can detect 12 copies of porcine epidemic diarrhea virus, 8 copies of porcine transmissible gastroenteritis virus, 24 copies of porcine rotavirus, 5 copies of porcine delta coronavirus, 18 copies of porcine bocavirus and 4 copies of porcine norovirus at the lowest.

Sequence listing

<110> Zhejiang agriculture and forestry university

<120> MLPA detection kit for detecting pathogenic nucleic acid of porcine viral diarrhea syndrome

<160> 26

<170> SIPOSequenceListing 1.0

<210> 1

<211> 17

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

gtctcggtgc agctgcg 17

<210> 2

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

gtagctgtgt tctgtctagc cagc 24

<210> 3

<211> 16

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

gccgtgttgg cagcag 16

<210> 4

<211> 28

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

tcatcatcac catagaagga gaagaggg 28

<210> 5

<211> 29

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

ctcaaaacaa aaaggctact catcctcag 29

<210> 6

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

gattgttggg gttgcgtttg g 21

<210> 7

<211> 34

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

ttgcaatctg ttaatgatta cctgtctttt agca 34

<210> 8

<211> 32

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

tgtccagaat cagatgtata ataaacacct gc 32

<210> 9

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

atgggaaata ggcaatccag cttg 24

<210> 10

<211> 50

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

gaaatggatt attgtattta ataagttcca attttagagg ttttctagac 50

<210> 11

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

atggccaacc agggacaac 19

<210> 12

<211> 27

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

tctttaaatt tggcatctgc atgaggt 27

<210> 13

<211> 49

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

gggttcccta agggttggac ctggctgggt ccagatatct gcactccga 49

<210> 14

<211> 53

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

gcgtagctca gtttgagtcc aacaagacct tctagattgg atcttgctgg cac 53

<210> 15

<211> 53

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 15

gggttcccta agggttggat caatgagggt cttccctctg gggtgccctg cgc 53

<210> 16

<211> 57

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

ctcccaatgg aactccatcg cccactggct cctctctaga ttggatcttg ctggcac 57

<210> 17

<211> 57

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 17

gggttcccta agggttggac ttaactccgc catcaaaccc gttgaaaacc atggcta 57

<210> 18

<211> 60

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 18

ctggctgcgt tacaccagac aaaagccagg tggcacttct agattggatc ttgctggcac 60

<210> 19

<211> 61

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 19

gggttcccta agggttggat ttcaattcac aaagggtgag ttgattactg gcacgcctaa 60

a 61

<210> 20

<211> 63

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 20

ccacttgaag gtgttacgga cgtttctttt atgactctgg tctagattgg atcttgctgg 60

cac 63

<210> 21

<211> 63

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 21

gggttcccta agggttggac ttccttcaaa gtgcatcttc tgctgaattc aaaaactacc 60

atc 63

<210> 22

<211> 68

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 22

taataattac tatcggcgct gccttgattg ctttgctatt cgctttctag attggatctt 60

gctggcac 68

<210> 23

<211> 83

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

gggttcccta agggttggat ggccaaccag ggacaacgtg tcagttgggg agatgaatct 60

accaaaacac gtggtcgttc caa 83

<210> 24

<211> 55

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 24

ttcccgtggt cggaagaata ataacatacc tctctagatt ggatcttgct ggcac 55

<210> 25

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 25

gggttcccta agggttgga 19

<210> 26

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 26

gtgccagcaa gatccaatct aga 23

Claims (3)

1. An MLPA detection kit for detecting pathogenic nucleic acid of porcine viral diarrhea syndrome, which is characterized by comprising:

(1) reverse transcription and pre-amplification mixture:

the mixture contains reverse transcription and pre-amplification primers, the sequences of which are shown as SEQ ID NO: 1-12, respectively used for reverse transcription and pre-amplification; wherein, the reverse transcription primer is a random primer with 12 basic groups;

(2) multiplex ligation probe mixture:

the mixed solution comprises a probe and a universal primer; wherein, the sequence of the probe is shown as SEQ ID NO: 13-24, and the sequence of the universal primer is shown as SEQ ID NO: 25-26;

(3) MLPA buffer solution;

(4) connecting buffer solutions A and B; wherein the ligation buffer A comprises Coenzyme NAD; the connection buffer solution B comprises Tris-HCl and MgCl₂、non-ionic detergent；

(5) Ligase Ligase-65;

(6) a PCR reaction mixed solution which comprises a universal primer shown in a sequence table SEQ ID NO. 25-26;

(7) SALSA polymerase;

(8) negative control;

(9) a positive control comprising a porcine epidemic diarrhea virus, a porcine transmissible gastroenteritis virus, a porcine rotavirus, a porcine delta coronavirus, a porcine bocavirus, and a porcine norovirus positive control plasmid;

the sequence of SEQ ID NO: 1-2 is a porcine bocavirus pre-amplification primer; the sequence of SEQ ID NO: 3-4 are porcine norovirus pre-amplification primers; the sequence of SEQ ID NO: 5-6 are porcine delta coronavirus pre-amplification primers; the sequence of SEQ ID NO: 7-8 is porcine epidemic diarrhea virus pre-amplification primer; the sequence of SEQ ID NO: 9-10 is a porcine rotavirus pre-amplification primer; the sequence of SEQ ID NO: 11-12 are porcine transmissible gastroenteritis virus pre-amplification primers; the sequence of SEQ ID NO: 13-14 are a left probe and a right probe for detecting the porcine bocavirus; the sequence of SEQ ID NO: 15-16 are left and right probes for detecting the porcine norovirus respectively; the sequence of SEQ ID NO: 17-18 are a left side probe and a right side probe for detecting the porcine delta coronavirus respectively; the sequence of SEQ ID NO: 19-20 are respectively a left side probe and a right side probe for detecting the porcine epidemic diarrhea virus; the sequence of SEQ ID NO: 21-22 are respectively a left side probe and a right side probe for detecting the porcine rotavirus; the sequence of SEQ ID NO: 23-24 are respectively a left side probe and a right side probe for detecting the transmissible gastroenteritis virus; wherein, the SEQ ID NO: 14. 16, 18, 20, 22 and 24' ends are subjected to phosphorylation treatment; the sequence of SEQ ID NO: 25. 26 are the forward and reverse primers, respectively, for universal PCR amplification.

2. The kit of claim 1, wherein the positive control in the kit is derived from a recombinant plasmid mixture obtained by reverse transcription of viral nucleic acid RNA and ligation of a specific gene fragment thereof to a plasmid; the specific genes are S gene of porcine epidemic diarrhea virus, N gene of porcine transmissible gastroenteritis virus, NSP1 gene of porcine rotavirus, N gene of porcine delta coronavirus, NS1 gene of porcine bocavirus and RdRp gene of porcine norovirus respectively.

3. The MLPA amplification detection method for detecting 6 porcine viral diarrhea syndrome pathogenic nucleic acids simultaneously by using the kit of claim 1, which is not used for diagnosis, and is characterized by comprising the following steps:

(1) extracting DNA and RNA of a sample by a magnetic bead method;

simultaneously extracting DNA and RNA in a sample by using a magnetic bead DNA and RNA co-extraction kit and a full-automatic nucleic acid extractor to obtain a 200 mu L sample;

(2) reverse transcription of RNA into cDNA and preamplification

Carrying out one-step reverse transcription RT-PCR reaction;

prepare 25 μ L reaction system: mu.L of OneStep Ahead RT-PCR Master Mix, 1. mu.L of OnStep Ahead RT-Mix, 5. mu.L of DNA or RNA, 1. mu.L of random reverse transcription primer and 5. mu.L of pre-amplification primer mixture, final concentration of 0.5. mu.M, 3. mu. L H per primer₂O is complemented;

reaction conditions are as follows: 10min at 50 ℃ and 5min at 95 ℃; at 95 ℃ for 15s, at 55 ℃ for 20s, at 72 ℃ for 20s, for 40 cycles; 2min at 72 ℃;

(3) MLPA amplification and detection

a) Denaturation of DNA

Taking 0.2mL PCR reaction tubes, adding 0.5 mu L DNA solution and 4.5 mu L TE into each tube, denaturing at 98 ℃ for 5min, and cooling to room temperature of 25 ℃;

b) hybridization of the Probe with sample DNA

Preparing 3 mu L of mixed probe liquid: 1.5. mu.L MLPA buffer solution + 1.5. mu.L probe mixture;

adding the probe mixture into the PCR reaction tube, incubating at 95 ℃ for 1min, hybridizing at 60 ℃ for 1-16h, and incubating at 54 ℃;

c) ligation of hybridization probes

Prepare 32 μ L ligase mix: 25 μ L dH₂O + 3. mu.L of a ligation buffer A + 3. mu.L of a ligation buffer B +1. mu.L of Ligase Ligase-65;

cooling the temperature of the PCR instrument to 54 ℃, opening a tube cover, adding 32 mu L of ligase mixture, incubating at 54 ℃ for 15min, heating at 98 ℃ for 5min to inactivate ligase, and incubating at 20 ℃;

d) PCR amplification of ligated probes

Prepare 10 μ L of PCR mix: 7.5 μ L dH₂O + 2. mu.L of PCR reaction mixture + 0.5. mu.L of SALSA polymerase;

taking out the PCR tube, and adding 10 mu L of PCR mixture at room temperature; the PCR reaction was started under the following conditions: 30s at 95 ℃, 30s at 60 ℃, 60s at 72 ℃ and 35 cycles; incubating at 72 deg.C for 20min, and cooling to 15 deg.C;

e) analysis by a full-automatic nucleic acid analyzer:

taking 20 mu L of PCR amplification product, and analyzing by using a full-automatic nucleic acid analyzer;

(4) result description and determination

a) Quality control standard:

the positive control has specific amplification bands at positions of 102bp, 110bp, 117bp, 124bp, 131bp and 138 bp;

negative control no specific amplification band;

if the negative control and the positive condition do not satisfy the above conditions, the test is regarded as invalid;

b) and (5) judging a result:

positive: a specific amplification band exists at a position of 102bp, which indicates that the porcine bocavirus exists in a sample; a specific amplification band exists at a position of 110bp, which indicates that the porcine norovirus exists in the sample; a specific amplification band exists at 117bp, which indicates that the porcine delta coronavirus exists in the sample; a specific amplification band exists at 124bp, which indicates that porcine epidemic diarrhea virus exists in the sample; a specific amplification strip is arranged at 131bp, which indicates that the porcine rotavirus exists in the sample; a specific amplification band exists at 138bp, which indicates that the transmissible gastroenteritis virus exists in the sample; meanwhile, MLPA amplification products can be sequenced and further confirmed;

negative: and (3) no specific amplification strip indicates that the sample does not contain porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus, porcine delta coronavirus, porcine bocavirus and porcine norovirus.

Images