CN113801951A - Primer probe set and kit for simultaneously detecting multiple pig-death pathogenic microorganisms and application of primer probe set and kit - Google Patents
Primer probe set and kit for simultaneously detecting multiple pig-death pathogenic microorganisms and application of primer probe set and kit Download PDFInfo
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Abstract
The invention relates to a diagnostic kit, in particular to a primer probe set and a kit for simultaneously detecting various pig death pathogenic microorganisms and application thereof. The invention provides a primer probe group for simultaneously detecting various porcine-lethal pathogenic microorganisms, wherein the nucleotide sequence of the primer probe group is shown as SEQ ID NO. 1-SEQ ID NO. 90. The primer probe set can be used for quickly, accurately and high-flux detecting pig morbidity or lethal pathogen. When the pathogeny of the pig is unknown or the general clinical characters such as digestive system diseases are known but the pathogeny is not clear, the primer probe set can be used for quickly locking whether the pathogeny which can cause the death of the pig is infected, so that the primer probe set is favorable for timely and accurate clinical medication and avoids more pig infection or death.
Description
Technical Field
The invention relates to a diagnostic kit, in particular to a primer probe set and a kit for simultaneously detecting various pig death pathogenic microorganisms and application thereof.
Background
The live pig breeding industry is an important component of domestic animal husbandry, and China is not only a live pig breeding country but also a pork consumption country. Therefore, the healthy and efficient breeding of live pigs plays an important role in live pig supply and pork safety. In the domestic live pig breeding industry, the situation of the pig diseases is more complex, the phenomena of mixed infection are more and more, the prevention and control of the swine plague of the swinery are more and more troublesome, and the economic loss caused by the live pig breeding industry every year can not be estimated. The method can quickly and definitely determine the pathogeny of the diseased pig to lock the pathogeny, is favorable for taking accurate prevention and intervention measures in time, effectively controls the spread of epidemic diseases in swinery, reduces the morbidity and mortality of the live pig, and is a necessary means for ensuring the healthy development of the live pig breeding industry.
However, in actual production, in the early stage of the disease of live pigs, clinical symptoms caused by different pathogenic infections are very similar, such as fever, anorexia, lassitude and the like, even if respiratory disease symptoms are all shown, the pathogens causing the infections cannot be clearly bacteria or viruses, and thus accurate medicine administration prevention and treatment cannot be realized. Therefore, there is a need to develop a method for rapidly, accurately and flux detecting pig pathogeny or lethal pathogen and effectively controlling the development of swine plague.
Disclosure of Invention
In order to solve the problems, the invention provides a primer probe set for simultaneously detecting a plurality of pig death pathogenic microorganisms, a kit and application thereof. The primer probe set can be used for rapidly, accurately and flux detecting pig morbidity or lethal pathogen.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a primer probe group for simultaneously detecting various porcine-lethal pathogenic microorganisms, wherein the nucleotide sequence of the primer probe group is shown as SEQ ID No. 1-SEQ ID No. 90.
Preferably, the pathogenic microorganisms include pathogenic microorganisms causing porcine digestive system diseases, pathogenic microorganisms causing porcine nervous system diseases, pathogenic microorganisms causing porcine respiratory system diseases, and pathogenic microorganisms causing porcine skin diseases.
Preferably, the pathogenic microorganisms causing the pig digestive system diseases comprise porcine epidemic diarrhea virus, porcine infectious gastroenteritis virus, rotavirus a, hog cholera virus, african hog cholera virus, salmonella cholera or paratyphoid, shiga toxin-producing escherichia coli in escherichia coli, enteropathogenic escherichia coli, enterotoxigenic escherichia coli, clostridium welchii and toxoplasma gondii; the pathogenic microorganisms causing the porcine nervous system diseases comprise rabies viruses, pseudorabies viruses, porcine encephalitis B viruses and listeria; the pathogenic microorganisms causing the porcine respiratory disease comprise porcine reproductive and respiratory syndrome virus, porcine circovirus, porcine influenza virus, porcine bacillus anthracis, porcine pasteurella multocida, haemophilus parasuis, streptococcus suis type 2, streptococcus suis type 9, mycoplasma pneumoniae and echinococcus; the pathogenic microorganisms causing the pig skin disease preferably comprise foot and mouth disease virus, erysipelothrix rhusiopathiae and eperythrozoon.
Preferably, the primer probe set for detecting the porcine epidemic diarrhea virus comprises PEDV-NF with a nucleotide sequence shown as SEQ ID No.1, PEDV-NR with a nucleotide shown as SEQ ID No.2 and PEDV-NP with a nucleotide shown as SEQ ID No. 3;
the primer probe group for detecting the porcine infectious gastroenteritis virus comprises TGEV-NF with a nucleotide sequence shown as SEQ ID No.4, TGEV-NR with a nucleotide sequence shown as SEQ ID No.5 and TGEV-NP with a nucleotide sequence shown as SEQ ID No. 6;
the primer probe set for detecting the A-type rotavirus comprises PRAV-VP6F with a nucleotide sequence shown as SEQ ID No.7, PRAV-VP6R with a nucleotide sequence shown as SEQ ID No.8 and PRAV-VP6P with a nucleotide sequence shown as SEQ ID No. 9;
the primer probe group for detecting the classical swine fever virus comprises CSFV-5UTRF with a nucleotide sequence shown as SEQ ID No.10, CSFV-5UTRR with a nucleotide sequence shown as SEQ ID No.11 and CSFV-5UTRP with a nucleotide sequence shown as SEQ ID No. 12;
the primer probe set for detecting African swine fever virus comprises ASFV-VP72F with a nucleotide sequence shown in SEQ ID No.13, ASFV-VP72R with a nucleotide sequence shown in SEQ ID No.14 and ASFV-VP72P with a nucleotide sequence shown in SEQ ID No. 15;
the primer probe group for detecting the cholera swine or the salmonella paratyphi comprises an STSP-hpF with a nucleotide sequence shown as SEQ ID No.16, an STSP-hpR with a nucleotide sequence shown as SEQ ID No.17 and an STSP-hpP with a nucleotide sequence shown as SEQ ID No.18
The primer probe group for detecting the shiga toxin-producing escherichia coli in the escherichia coli comprises STEC-stx1F1 with a nucleotide sequence shown as SEQ ID No.19, STEC-stx1R1 with a nucleotide sequence shown as SEQ ID No.20, STEC-stx1P1 with a nucleotide sequence shown as SEQ ID No.21, STEC-stx2F2 with a nucleotide sequence shown as SEQ ID No.22, STEC-stx2R2 with a nucleotide sequence shown as SEQ ID No.23 and STEC-stx2P2 with a nucleotide sequence shown as SEQ ID No. 24;
the primer probe group for detecting the enteropathogenic escherichia coli comprises EPEC-eaeF with a nucleotide sequence shown as SEQ ID No.25, EPEC-eaeR with a nucleotide sequence shown as SEQ ID No.26 and EPEC-eaeP with a nucleotide sequence shown as SEQ ID No. 27;
the primer probe set for detecting enterotoxigenic escherichia coli comprises ETEC-eltF1 with a nucleotide sequence shown as SEQ ID No.28, ETEC-eltR1 with a nucleotide sequence shown as SEQ ID No.29, EPEC-eae P1 with a nucleotide sequence shown as SEQ ID No.30, ETEC-estpF2 with a nucleotide sequence shown as SEQ ID No.31, ETEC-estpR2 with a nucleotide sequence shown as SEQ ID No.32 and ETEC-estpP2 with a nucleotide sequence shown as SEQ ID No. 33;
the primer probe group for detecting the clostridium welchii comprises Cp-cpaF with a nucleotide sequence shown as SEQ ID No.34, Cp-cpaR with a nucleotide sequence shown as SEQ ID No.35 and Cp-cpaP with a nucleotide sequence shown as SEQ ID No. 36;
the primer probe group for detecting the toxoplasma comprises TG-B1F with a nucleotide sequence shown as SEQ ID No.37, TG-B1R with a nucleotide sequence shown as SEQ ID No.38 and TG-B1P with a nucleotide sequence shown as SEQ ID No. 39;
the primer probe group for detecting the rabies virus comprises RV-NF with a nucleotide sequence shown as SEQ ID No.40, RV-NR with a nucleotide sequence shown as SEQ ID No.41 and RV-NP with a nucleotide sequence shown as SEQ ID No. 42;
the primer probe set for detecting the pseudorabies virus comprises PRV-gEF with a nucleotide sequence shown as SEQ ID No.43, PRV-gER with a nucleotide sequence shown as SEQ ID No.44 and PRV-gEP with a nucleotide sequence shown as SEQ ID No. 45;
the primer probe group for detecting the porcine encephalitis B virus comprises JEV-NSF with a nucleotide sequence shown as SEQ ID No.46, JEV-NSR with a nucleotide sequence shown as SEQ ID No.47 and JEV-NSP with a nucleotide sequence shown as SEQ ID No. 48;
the primer probe set for detecting the listeria comprises Li-F with a nucleotide sequence shown as SEQ ID No.49, Li-R with a nucleotide sequence shown as SEQ ID No.50 and Li-P with a nucleotide sequence shown as SEQ ID No. 51;
the primer probe set for detecting the porcine reproductive and respiratory syndrome virus comprises PRRSV-NSP2F with a nucleotide sequence shown as SEQ ID No.52, PRRSV-NSP2R with a nucleotide sequence shown as SEQ ID No.53 and PRRSV-NSP2P with a nucleotide sequence shown as SEQ ID No. 54;
the primer probe set for detecting the porcine circovirus comprises PCV-ORF2F with a nucleotide sequence shown as SEQ ID No.55, PCV-ORF2R with a nucleotide sequence shown as SEQ ID No.56 and PCV-ORF2P with a nucleotide sequence shown as SEQ ID No. 57;
the primer probe set for detecting the swine influenza virus comprises SIV-MF with a nucleotide sequence shown as SEQ ID No.58, SIV-MR with a nucleotide sequence shown as SEQ ID No.59 and SIV-MP with a nucleotide sequence shown as SEQ ID No. 60;
the primer probe set for detecting the swine bacillus anthracis comprises BA-plcRF with a nucleotide sequence shown as SEQ ID No.61, BA-plcRR with a nucleotide sequence shown as SEQ ID No.62 and BA-plcRP with a nucleotide sequence shown as SEQ ID No. 63;
the primer probe set for detecting the swine pasteurella multocida comprises Pm-SodAF with a nucleotide sequence shown as SEQ ID No.64, Pm-SodAR with a nucleotide sequence shown as SEQ ID No.65 and Pm-SodAP with a nucleotide sequence shown as SEQ ID No. 66;
the primer probe set for detecting the haemophilus parasuis comprises HP-INFBF with a nucleotide sequence shown as SEQ ID No.67, HP-INFBR with a nucleotide sequence shown as SEQ ID No.68 and HP-INFBP with a nucleotide sequence shown as SEQ ID No. 69;
the primer probe set for detecting the streptococcus suis type 2 comprises SS2-CPS2JF with a nucleotide sequence shown as SEQ ID No.70, SS2-CPS2JR with a nucleotide sequence shown as SEQ ID No.71 and SS2-CPS2JP with a nucleotide sequence shown as SEQ ID No. 72;
the primer probe set for detecting the streptococcus suis 9 type comprises SS9-CPS9HF with a nucleotide sequence shown as SEQ ID No.73, SS9-CPS9HR with a nucleotide sequence shown as SEQ ID No.74 and SS9-CPS9HP with a nucleotide sequence shown as SEQ ID No. 75;
the primer probe set for detecting the mycoplasma pneumoniae comprises MHP-p102F with a nucleotide sequence shown as SEQ ID No.76, MHP-p102R with a nucleotide sequence shown as SEQ ID No.77 and MHP-p102P with a nucleotide sequence shown as SEQ ID No. 78;
the primer probe set for detecting echinococcus comprises EG/M-12sF with a nucleotide sequence shown as SEQ ID No.79, EG/M-12sR with a nucleotide sequence shown as SEQ ID No.80 and EG/M-12sP with a nucleotide sequence shown as SEQ ID No. 81;
the primer probe group for detecting the foot-and-mouth disease virus comprises FMDV-3DF with a nucleotide sequence shown as SEQ ID No.82, FMDV-3DR with a nucleotide sequence shown as SEQ ID No.83 and FMDV-3DP with a nucleotide sequence shown as SEQ ID No. 84;
the primer probe set for detecting the erysipelothrix rhusiopathiae comprises ER-16sF with a nucleotide sequence shown as SEQ ID No.85, ER-16sR with a nucleotide sequence shown as SEQ ID No.86 and ER-16sP with a nucleotide sequence shown as SEQ ID No. 87;
the primer probe set for detecting the eperythrozoon comprises Ms-16sF with a nucleotide sequence shown as SEQ ID No.88, Ms-16sR with a nucleotide sequence shown as SEQ ID No.89 and Ms-16sP with a nucleotide sequence shown as SEQ ID No. 90.
The invention provides a kit for simultaneously detecting various porcine-lethal pathogenic microorganisms, which comprises the primer probe set.
Preferably, the working concentration of each primer in the primer probe set is 0.5. mu. mol/L, and the working concentration of each probe is 0.05. mu. mol/L.
Preferably, the kit also comprises Real time PCR buffer solution, Taq enzyme, dNTP mixed solution and MgCl2(ii) a The working concentration of the Taq enzyme is 0.03-0.1U/mu L; the working concentration of the dNTP mixed solution is 0.1-0.4 mmol/L, and MgCl is added2The working concentration of (a) is 2-6 mmol/L.
The invention provides application of the primer probe set or the kit in non-diagnosis purpose detection of various pig death pathogenic microorganisms.
Preferably, in the detection, the reaction system of the PCR is calculated by 25 mu L, and comprises 0.5-1 mu L of DNA template or cDNA template and TaKaRa TB GreenTMPremix Ex TaqTMII 12.5. mu.L of each primer probe set for each pathogenic microorganism 10. mu.L and the balance ddH2O。
Preferably, the PCR amplification procedure is as follows: pre-denaturation at 95 ℃ for 15 min; denaturation at 95 ℃ for 15s, annealing and extension at 58 ℃ for 1min, and 45 cycles; the temperature is reduced to 4 ℃.
Has the advantages that: the invention provides a primer probe group for simultaneously detecting various pig death pathogenic microorganisms, wherein the nucleotide sequence of the primer group of the probe is shown in SEQ ID No. 1-SEQ ID No.90, and the primer probe group can be used for quickly, accurately and high-flux detecting pig morbidity or lethal pathogeny. When the pathogeny of the pig morbidity or mortality is unknown, or the general clinical characters such as digestive system diseases are known but the pathogeny is not clear, the primer probe set can be used for quickly locking whether the pathogeny which can cause the pig mortality is infected, so that timely and accurate clinical medication is facilitated, and more pig infections or deaths are avoided.
Moreover, the invention provides a kit for simultaneously detecting a plurality of pig-death pathogenic microorganisms. The kit has the characteristics of rapidness, high flux, specificity, sensitivity, simple operation, easy interpretation and the like, can be freely and flexibly combined according to different types of clinical symptoms such as symptoms of a nervous system, a digestive system, a respiratory system, skin and the like, and is more economical and practical.
Drawings
FIG. 1 is a diagram showing the detection of common pathogenic microorganisms causing swine bacterial diseases by using a specific primer probe set in a kit, wherein, Delta Rn represents the amount of probe degradation in the PCR process, namely the amount of PCR products, 2 is the positive detection of Listeria, 3 is the positive detection of Pasteurella multocida, 5 is the positive detection (est) of enterotoxigenic Escherichia coli, 7 is the positive detection of enteropathogenic Escherichia coli, 11 is the positive detection (stx1) of Shiga toxigenic Escherichia coli, and 12 is the positive detection (stx2) of Shiga toxigenic Escherichia coli; 15 is positive detection of salmonella choleraesuis, 16 is positive detection of haemophilus parasuis, 18 is positive detection (elt) of enterotoxigenic escherichia coli, 22 is positive detection of clostridium welchii, and 41 is treatment without shiga toxin-producing escherichia coli template, namely negative control.
Detailed Description
The reagent of the present invention is obtained by ordinary purchase by those skilled in the art, unless otherwise specified.
The invention provides a primer probe group for simultaneously detecting various porcine-lethal pathogenic microorganisms, wherein the nucleotide sequence of the primer group of the probe is shown as SEQ ID No. 1-SEQ ID No. 90. In the present invention, the pathogenic microorganisms preferably include pathogenic microorganisms inducing porcine digestive system diseases, pathogenic microorganisms inducing porcine nervous system diseases, pathogenic microorganisms inducing porcine respiratory system diseases, and pathogenic microorganisms inducing porcine skin diseases; the pathogenic microorganisms causing the pig digestive system diseases preferably comprise porcine epidemic diarrhea virus, porcine infectious gastroenteritis virus, A-type rotavirus, classical swine fever virus, African swine fever virus, hog cholera or paratyphoid salmonella, shiga toxin-producing escherichia coli, enteropathogenic escherichia coli, enterotoxigenic escherichia coli, clostridium welchii and toxoplasma gondii; the pathogenic microorganisms causing the porcine nervous system diseases preferably comprise rabies viruses, pseudorabies viruses, porcine encephalitis B viruses and listeria; the pathogenic microorganisms causing the porcine respiratory disease preferably comprise porcine reproductive and respiratory syndrome virus, porcine circovirus, porcine influenza virus, porcine bacillus anthracis, porcine pasteurella multocida, haemophilus parasuis, streptococcus suis type 2, streptococcus suis type 9, mycoplasma pneumoniae and echinococcus; preferably, the pathogenic microorganisms inducing swine skin disease include foot and mouth disease virus, erysipelothrix rhusiopathiae and eperythrozoon.
In the present invention, the specific information of the primer probe set is preferably as shown in table 1; the primer probe set for detecting the porcine epidemic diarrhea virus is preferably designed based on the N gene specificity of the porcine epidemic diarrhea virus; the primer probe group for detecting the porcine infectious gastroenteritis virus is preferably designed based on the N gene specificity of the porcine infectious gastroenteritis virus; the primer probe set for detecting the type a rotavirus is preferably designed based on the VP6 gene specificity of the type a rotavirus; the primer probe set for detecting the classical swine fever virus is preferably designed aiming at the 5UTR gene specificity of the classical swine fever virus; the primer probe set for detecting the African swine fever virus is preferably designed based on the VP72 gene specificity of the African swine fever virus; the primer probe group for detecting the cholera suis or the salmonella paratyphi is preferably designed based on the hp gene specificity of the cholera suis or the salmonella paratyphi; the primer probe set for detecting the shiga toxin-producing escherichia coli in the escherichia coli is preferably designed based on the stx1 and stx2 gene specificities of the shiga toxin-producing escherichia coli in the escherichia coli; the primer probe set for detecting enteropathogenic escherichia coli is preferably designed based on the eae gene specificity of enteropathogenic escherichia coli; the primer probe set for detecting enterotoxigenic escherichia coli is preferably designed based on the elt and est gene specificities of enterotoxigenic escherichia coli; the primer probe set for detecting clostridium welchii is preferably designed based on the cpa gene specificity of clostridium welchii; the primer probe set for detecting the toxoplasma is preferably designed based on the B1 gene specificity of the toxoplasma; the primer probe set for detecting the rabies virus is preferably designed based on the N gene specificity of the rabies virus; the primer probe group for detecting the pseudorabies virus is preferably designed based on the gE gene specificity of the pseudorabies virus; the primer probe group for detecting the porcine encephalitis B virus is preferably designed based on the NS gene specificity of the porcine encephalitis B virus; the primer probe set for detecting listeria is preferably designed based on the h1y gene specificity of listeria; the primer probe set for detecting the porcine reproductive and respiratory syndrome virus is preferably designed based on the NSP2 gene specificity of the porcine reproductive and respiratory syndrome virus; the primer probe set for detecting the porcine circovirus is preferably designed based on the ORF2 gene specificity of the porcine circovirus; the primer probe set for detecting the swine influenza virus is preferably designed based on the M gene specificity of the swine influenza virus; the optimization of the primer probe group for detecting the swine bacillus anthracis is designed based on the specificity of the plcR gene of the swine bacillus anthracis; the primer probe set for detecting the swine pasteurella multocida is preferably designed based on the SodA gene specificity of the swine pasteurella multocida; the primer probe group for detecting the haemophilus parasuis is preferably designed based on the infB gene specificity of the haemophilus parasuis; the primer probe set for detecting the streptococcus suis type 2 is preferably designed based on the cps2J gene specificity of the streptococcus suis type 2; the primer probe set for detecting the streptococcus suis type 9 is preferably designed based on the cps9H gene specificity of the streptococcus suis type 9; the primer probe group for detecting the mycoplasma pneumoniae is preferably designed based on the p102 gene specificity of the mycoplasma pneumoniae; the primer probe set for detecting echinococcosis is preferably designed based on the specificity of 12srRNA for echinococcosis; the primer probe group for detecting the foot-and-mouth disease virus is preferably designed based on the 3D gene specificity of the foot-and-mouth disease virus; the primer probe set for detecting erysipelothrix rhusiopathiae is preferably designed based on the 16srRNA specificity of erysipelothrix rhusiopathiae; the primer probe set for detecting the eperythrozoon is preferably designed based on the 16 srna specificity of the eperythrozoon.
TABLE 1 detailed information of primer Probe sets
The invention provides a kit for simultaneously detecting multiple pig death pathogenic microorganisms, which comprises the primer probe group. In the invention, the working concentration of each primer in the primer probe set is preferably 0.2-1 μmol/L, more preferably 0.5 μmol/L, and the working concentration of each probe is preferably 0.02-0.2 μmol-L, more preferably 0.05. mu. mol/L; the kit preferably further comprises Real time PCR buffer solution, Taq enzyme, dNTP mixed solution and MgCl2(ii) a The Real time PCR buffer solution, Taq enzyme, dNTP mixed solution and MgCl2Preferably TaKaRa TB GreenTMPremix Ex TaqTMII (TaKaRa, RR 820); the working concentration of the Taq enzyme is preferably 0.03-0.1U/mu L, and more preferably 0.06-0.09U/mu L; the working concentration of the dNTP mixed solution is preferably 0.1-0.4 mmol/L, and more preferably 0.2-0.3 mmol/L; said MgCl2The working concentration of (A) is preferably 2 to 6mmol/L, more preferably 3 to 5mmol/L, and most preferably 3.5 to 4.5 mmol/L. The kit of the present invention is preferably a kit of parts.
When the pathogeny of the pig morbidity or mortality is unknown, or the general clinical characters such as digestive system diseases are known but the pathogeny is not clear, the primer probe set can be used for quickly locking whether the pathogeny which can cause the pig mortality is infected, so that timely and accurate clinical medication is facilitated, and more pig infections or deaths are avoided. Therefore, the primer probe set or the kit containing the primer probe set can be used for detecting various pig death pathogenic microorganisms.
The invention provides application of the primer probe set or the kit in non-diagnosis purpose detection of various porcine death pathogenic microorganisms. In the detection of the invention, the reaction system of PCR is 25 muL, preferably comprises 0.5-1 muL of DNA template or cDNA template and TaKaRa TB GreenTMPremix Ex TaqTMII 12.5. mu.L of each primer probe set for each pathogenic microorganism 10. mu.L and the balance ddH2O; the PCR amplification procedure is preferably: pre-denaturation at 95 ℃ for 15 min; denaturation at 95 ℃ for 15s, annealing and extension at 58 ℃ for 1min, and 45 cycles; the temperature is reduced to 4 ℃. The kit has the characteristics of rapidness, flux, specificity, sensitivity, simple operation, easy interpretation and the like, can be freely and flexibly combined according to different types of clinical symptoms such as symptoms of a nervous system, a digestive system, a respiratory system, skin and the like, and is more economical and practical.
In order to further illustrate the present invention, the primer probe set, the kit and the application thereof for simultaneously detecting a plurality of pig-death pathogenic microorganisms provided by the present invention are described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.
Example 1
Common bacteria (Listeria, Pasteurella multocida, enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli, Shiga toxin-producing Escherichia coli, hog cholera or salmonella paratyphi, haemophilus parasuis and Clostridium welchii) capable of causing bacterial diseases of live pigs are selected, and the specific primer probe sets in the kit are used for detection so as to verify the effectiveness of the primer probe sets. Preparing a RealtimePCR reaction system: each reaction tube contains 1 mu L of template DNA of different bacteria (Listeria, Pasteurella multocida, enterotoxigenic Escherichia coli, Shiga toxin-producing Escherichia coli, enteropathogenic Escherichia coli, hog cholera or paratyphoid salmonella, haemophilus parasuis and clostridium welchii), and TaKaRa TB GreenTMPremix Ex TaqTMII (TaKaRa, RR820) 12.5. mu.L and 10. mu.L each of the primer probe sets for each bacterium, ddH2Make up to 25. mu.L of O. Two specific genes of enterotoxigenic escherichia coli and shiga toxin-producing escherichia coli are detected respectively, and Neg (without adding a shiga toxin-producing escherichia coli template) is used as a negative control. Performing PCR detection by a Realtime PCR instrument, wherein the PCR amplification reaction comprises the following steps: pre-denaturation at 95 ℃ for 15 min; denaturation at 95 ℃ for 15s, annealing and extension at 58 ℃ for 1min, and 45 cycles; the temperature is reduced to 4 ℃. Interpreting the detection data, wherein the detection data is shown in FIG. 1: the 10 probe primer groups tested were found to all have positive amplification to their specific strain template DNA, while the negative control for listeria primers without template did not have positive amplification.
Application example 1
The etiology diagnosis is carried out on pigs with unknown etiology or death in pig farms
Simultaneously extracting DNA and RNA of diseased or dead pig focus part tissues or immune organs by adopting a DNA/RNA co-extraction kit (Tiangen, DP422), wherein the RNA is reversely transcribed into cDNA by adopting a Fastking one-step reverse transcription kit (Tiangen, KR118), and then the kit is used for simultaneously detecting various porcine pathogensA kit for detecting nucleic acid from a clinical sample of a killed pathogenic microorganism wherein the RNA virus utilises a reverse transcribed cDNA template and the remainder utilises an extracted DNA template. Preparing a fluorescent quantitative PCR reaction system (DNA template or cDNA template 1 mu L, TaKaRa TB Green)TMPremix Ex TaqTMII (TaKaRa, RR820) 12.5. mu.L and primer Probe set 10. mu.L, ddH2O to 25 μ L), performing PCR detection by a realtome PCR instrument, and performing PCR amplification reaction as follows: pre-denaturation at 95 ℃ for 15 min; denaturation at 95 ℃ for 15s, annealing and extension at 58 ℃ for 1min, and 45 cycles; cooling to 4 ℃; then, the detection data is interpreted, and the African swine fever virus corresponding to the amplified positive gene is the pathogen causing the pig morbidity or mortality.
Application example 2
Pathogen detection of suspected dead or diseased pig carcasses
The method comprises the steps of detecting abnormal sensory pig carcass, namely dead or diseased pigs suspected to be slaughtered, simultaneously extracting DNA and RNA of focus part tissues or immune organs of the pig carcass by adopting a test DNA/RNA co-extraction kit (Tiangen, DP422), wherein the RNA is reversely transcribed into cDNA by adopting a Fastking one-step reverse transcription kit (Tiangen, KR118), and then detecting nucleic acid of clinical samples by utilizing the kit for simultaneously detecting various pig death pathogenic microorganisms, wherein RNA virus utilizes a reverse transcribed cDNA template, and the rest utilizes the extracted DNA template. Preparing a fluorescent quantitative PCR reaction system (DNA template or cDNA template 1 mu L, TaKaRa TB Green)TMPremix Ex TaqTMII (TaKaRa, RR820) 12.5. mu.L and primer Probe set 10. mu.L, ddH2O to 25 μ L), performing PCR detection by a realtome PCR instrument, and performing PCR amplification reaction as follows: pre-denaturation at 95 ℃ for 15 min; denaturation at 95 ℃ for 15s, annealing and extension at 58 ℃ for 1min, and 45 cycles; cooling to 4 ℃; then the detection data is interpreted, and the clostridium welchii corresponding to the positive amplification gene is the pathogen causing the death of the pig.
Application example 3
Etiological diagnosis of swine with digestive system diseases
Extracting digestive system clinical symptoms by using kit DNA/RNA co-extraction kit (Tiangen, DP422)The RNA is reversely transcribed into cDNA by adopting a Fastking one-step reverse transcription kit (Tiangen, KR118), then the nucleic acid of clinical samples is detected by using the primer probe for detecting 11 types of digestive system pathogens contained in the kit for simultaneously detecting various types of pathogenic microorganisms causing pig death, and meanwhile, positive controls (the positive nucleic acid of porcine epidemic diarrhea virus, porcine infectious gastroenteritis virus, A-type rotavirus, classical swine fever virus, African swine fever virus, swine cholera or salmonella paratyphi, shiga toxin-producing escherichia coli, enteropathogenic escherichia coli, enterotoxigenic escherichia coli, clostridium welchii and 11 types of pathogenic microorganisms of toxoplasma are) and negative controls (no template is added) are made, wherein the RNA virus utilizes a reverse transcription cDNA template, and the rest utilizes a DNA template. Preparing a fluorescent quantitative PCR reaction system (DNA template or cDNA template 1 mu L, TaKaRa TB Green)TMPremix Ex TaqTMII (TaKaRa, RR820) 12.5. mu.L and primer Probe set 10. mu.L, ddH2O to 25 μ L), performing PCR detection by a realtome PCR instrument, and performing PCR amplification reaction as follows: pre-denaturation at 95 ℃ for 15 min; denaturation at 95 ℃ for 15s, annealing and extension at 58 ℃ for 1min, and 45 cycles; cooling to 4 ℃; then the detection data is interpreted, and the shiga toxin-producing escherichia coli corresponding to the positive amplification gene is the pathogen causing the digestive system diseases of the pigs.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> China center for animal health and epidemiology
<120> primer probe set and kit for simultaneously detecting multiple pig death pathogenic microorganisms and application thereof
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<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
Claims (10)
1. A primer probe group for simultaneously detecting various porcine-lethal pathogenic microorganisms is characterized in that the nucleotide sequence of the primer probe group is shown as SEQ ID No. 1-SEQ ID No. 90.
2. The primer probe set of claim 1, wherein the pathogenic microorganisms comprise pathogenic microorganisms causing porcine digestive system diseases, pathogenic microorganisms causing porcine nervous system diseases, pathogenic microorganisms causing porcine respiratory system diseases, preferably pathogenic microorganisms causing porcine skin diseases.
3. The primer-probe set of claim 2, wherein the pathogenic microorganisms causing digestive system disease in pigs comprise porcine epidemic diarrhea virus, porcine infectious gastroenteritis virus, rotavirus a, classical swine fever virus, african swine fever virus, salmonella cholera or paratyphoid, shiga toxin-producing escherichia coli, enteropathogenic escherichia coli, enterotoxigenic escherichia coli, clostridium welchii and toxoplasma gondii; the pathogenic microorganisms causing the porcine nervous system diseases comprise rabies viruses, pseudorabies viruses, porcine encephalitis B viruses and listeria; the pathogenic microorganisms causing the porcine respiratory disease comprise porcine reproductive and respiratory syndrome virus, porcine circovirus, porcine influenza virus, porcine bacillus anthracis, porcine pasteurella multocida, haemophilus parasuis, streptococcus suis type 2, streptococcus suis type 9, mycoplasma pneumoniae and echinococcus; the pathogenic microorganisms causing the pig skin disease preferably comprise foot and mouth disease virus, erysipelothrix rhusiopathiae and eperythrozoon.
4. The primer probe set according to any one of claims 1 to 3, wherein the primer probe set for detecting porcine epidemic diarrhea virus comprises PEDV-NF with a nucleotide sequence shown as SEQ ID No.1, PEDV-NR with a nucleotide shown as SEQ ID No.2 and PEDV-NP with a nucleotide shown as SEQ ID No. 3;
the primer probe group for detecting the porcine infectious gastroenteritis virus comprises TGEV-NF with a nucleotide sequence shown as SEQ ID No.4, TGEV-NR with a nucleotide sequence shown as SEQ ID No.5 and TGEV-NP with a nucleotide sequence shown as SEQ ID No. 6;
the primer probe set for detecting the A-type rotavirus comprises PRAV-VP6F with a nucleotide sequence shown as SEQ ID No.7, PRAV-VP6R with a nucleotide sequence shown as SEQ ID No.8 and PRAV-VP6P with a nucleotide sequence shown as SEQ ID No. 9;
the primer probe group for detecting the classical swine fever virus comprises CSFV-5UTRF with a nucleotide sequence shown as SEQ ID No.10, CSFV-5UTRR with a nucleotide sequence shown as SEQ ID No.11 and CSFV-5UTRP with a nucleotide sequence shown as SEQ ID No. 12;
the primer probe set for detecting African swine fever virus comprises ASFV-VP72F with a nucleotide sequence shown in SEQ ID No.13, ASFV-VP72R with a nucleotide sequence shown in SEQ ID No.14 and ASFV-VP72P with a nucleotide sequence shown in SEQ ID No. 15;
the primer probe group for detecting the cholera swine or the salmonella paratyphi comprises an STSP-hpF with a nucleotide sequence shown as SEQ ID No.16, an STSP-hpR with a nucleotide sequence shown as SEQ ID No.17 and an STSP-hpP with a nucleotide sequence shown as SEQ ID No.18
The primer probe group for detecting the shiga toxin-producing escherichia coli in the escherichia coli comprises STEC-stx1F1 with a nucleotide sequence shown as SEQ ID No.19, STEC-stx1R1 with a nucleotide sequence shown as SEQ ID No.20, STEC-stx1P1 with a nucleotide sequence shown as SEQ ID No.21, STEC-stx2F2 with a nucleotide sequence shown as SEQ ID No.22, STEC-stx2R2 with a nucleotide sequence shown as SEQ ID No.23 and STEC-stx2P2 with a nucleotide sequence shown as SEQ ID No. 24;
the primer probe group for detecting the enteropathogenic escherichia coli comprises EPEC-eaeF with a nucleotide sequence shown as SEQ ID No.25, EPEC-eaeR with a nucleotide sequence shown as SEQ ID No.26 and EPEC-eaeP with a nucleotide sequence shown as SEQ ID No. 27;
the primer probe set for detecting enterotoxigenic escherichia coli comprises ETEC-eltF1 with a nucleotide sequence shown as SEQ ID No.28, ETEC-eltR1 with a nucleotide sequence shown as SEQ ID No.29, EPEC-eae P1 with a nucleotide sequence shown as SEQ ID No.30, ETEC-estpF2 with a nucleotide sequence shown as SEQ ID No.31, ETEC-estpR2 with a nucleotide sequence shown as SEQ ID No.32 and ETEC-estpP2 with a nucleotide sequence shown as SEQ ID No. 33;
the primer probe group for detecting the clostridium welchii comprises Cp-cpaF with a nucleotide sequence shown as SEQ ID No.34, Cp-cpaR with a nucleotide sequence shown as SEQ ID No.35 and Cp-cpaP with a nucleotide sequence shown as SEQ ID No. 36;
the primer probe group for detecting the toxoplasma comprises TG-B1F with a nucleotide sequence shown as SEQ ID No.37, TG-B1R with a nucleotide sequence shown as SEQ ID No.38 and TG-B1P with a nucleotide sequence shown as SEQ ID No. 39;
the primer probe group for detecting the rabies virus comprises RV-NF with a nucleotide sequence shown as SEQ ID No.40, RV-NR with a nucleotide sequence shown as SEQ ID No.41 and RV-NP with a nucleotide sequence shown as SEQ ID No. 42;
the primer probe set for detecting the pseudorabies virus comprises PRV-gEF with a nucleotide sequence shown as SEQ ID No.43, PRV-gER with a nucleotide sequence shown as SEQ ID No.44 and PRV-gEP with a nucleotide sequence shown as SEQ ID No. 45;
the primer probe group for detecting the porcine encephalitis B virus comprises JEV-NSF with a nucleotide sequence shown as SEQ ID No.46, JEV-NSR with a nucleotide sequence shown as SEQ ID No.47 and JEV-NSP with a nucleotide sequence shown as SEQ ID No. 48;
the primer probe set for detecting the listeria comprises Li-F with a nucleotide sequence shown as SEQ ID No.49, Li-R with a nucleotide sequence shown as SEQ ID No.50 and Li-P with a nucleotide sequence shown as SEQ ID No. 51;
the primer probe set for detecting the porcine reproductive and respiratory syndrome virus comprises PRRSV-NSP2F with a nucleotide sequence shown as SEQ ID No.52, PRRSV-NSP2R with a nucleotide sequence shown as SEQ ID No.53 and PRRSV-NSP2P with a nucleotide sequence shown as SEQ ID No. 54;
the primer probe set for detecting the porcine circovirus comprises PCV-ORF2F with a nucleotide sequence shown as SEQ ID No.55, PCV-ORF2R with a nucleotide sequence shown as SEQ ID No.56 and PCV-ORF2P with a nucleotide sequence shown as SEQ ID No. 57;
the primer probe set for detecting the swine influenza virus comprises SIV-MF with a nucleotide sequence shown as SEQ ID No.58, SIV-MR with a nucleotide sequence shown as SEQ ID No.59 and SIV-MP with a nucleotide sequence shown as SEQ ID No. 60;
the primer probe set for detecting the swine bacillus anthracis comprises BA-plcRF with a nucleotide sequence shown as SEQ ID No.61, BA-plcRR with a nucleotide sequence shown as SEQ ID No.62 and BA-plcRP with a nucleotide sequence shown as SEQ ID No. 63;
the primer probe set for detecting the swine pasteurella multocida comprises Pm-SodAF with a nucleotide sequence shown as SEQ ID No.64, Pm-SodAR with a nucleotide sequence shown as SEQ ID No.65 and Pm-SodAP with a nucleotide sequence shown as SEQ ID No. 66;
the primer probe set for detecting the haemophilus parasuis comprises HP-INFBF with a nucleotide sequence shown as SEQ ID No.67, HP-INFBR with a nucleotide sequence shown as SEQ ID No.68 and HP-INFBP with a nucleotide sequence shown as SEQ ID No. 69;
the primer probe set for detecting the streptococcus suis type 2 comprises SS2-CPS2JF with a nucleotide sequence shown as SEQ ID No.70, SS2-CPS2JR with a nucleotide sequence shown as SEQ ID No.71 and SS2-CPS2JP with a nucleotide sequence shown as SEQ ID No. 72;
the primer probe set for detecting the streptococcus suis 9 type comprises SS9-CPS9HF with a nucleotide sequence shown as SEQ ID No.73, SS9-CPS9HR with a nucleotide sequence shown as SEQ ID No.74 and SS9-CPS9HP with a nucleotide sequence shown as SEQ ID No. 75;
the primer probe set for detecting the mycoplasma pneumoniae comprises MHP-p102F with a nucleotide sequence shown as SEQ ID No.76, MHP-p102R with a nucleotide sequence shown as SEQ ID No.77 and MHP-p102P with a nucleotide sequence shown as SEQ ID No. 78;
the primer probe set for detecting echinococcus comprises EG/M-12sF with a nucleotide sequence shown as SEQ ID No.79, EG/M-12sR with a nucleotide sequence shown as SEQ ID No.80 and EG/M-12sP with a nucleotide sequence shown as SEQ ID No. 81;
the primer probe group for detecting the foot-and-mouth disease virus comprises FMDV-3DF with a nucleotide sequence shown as SEQ ID No.82, FMDV-3DR with a nucleotide sequence shown as SEQ ID No.83 and FMDV-3DP with a nucleotide sequence shown as SEQ ID No. 84;
the primer probe set for detecting the erysipelothrix rhusiopathiae comprises ER-16sF with a nucleotide sequence shown as SEQ ID No.85, ER-16sR with a nucleotide sequence shown as SEQ ID No.86 and ER-16sP with a nucleotide sequence shown as SEQ ID No. 87;
the primer probe set for detecting the eperythrozoon comprises Ms-16sF with a nucleotide sequence shown as SEQ ID No.88, Ms-16sR with a nucleotide sequence shown as SEQ ID No.89 and Ms-16sP with a nucleotide sequence shown as SEQ ID No. 90.
5. A kit for simultaneously detecting a plurality of pig-death pathogenic microorganisms, wherein the kit comprises the primer probe set of any one of claims 1 to 4.
6. The kit of claim 5, wherein each primer in the primer probe set has a working concentration of 0.2-1 μmol/L, and each probe has a working concentration of 0.02-0.2 μmol/L.
7. The kit of claim 5, wherein the kit further comprises Real time PCR buffer, Taq enzyme, dNTP mix and MgCl2(ii) a The working concentration of the Taq enzyme is 0.03-0.1U/mu L; the working concentration of the dNTP mixed solution is 0.1-0.4 mmol/L, and MgCl is added2The working concentration of (a) is 2-6 mmol/L.
8. Use of the primer probe set of any one of claims 1 to 4 or the kit of any one of claims 5 to 7 for the detection of a plurality of porcine-deaths causing pathogenic microorganisms for non-diagnostic purposes.
9. The application of claim 8, wherein in the detection, the reaction system of the PCR is 25 μ L, and comprises 0.5-1 μ L of DNA template or cDNA template, and TaKaRa TB GreenTMPremix Ex TaqTMII 12.5. mu.L of each primer probe set for each pathogenic microorganism 10. mu.L and the balance ddH2O。
10. The use of claim 9, wherein the PCR amplification procedure is: pre-denaturation at 95 ℃ for 15 min; denaturation at 95 ℃ for 15s, annealing and extension at 58 ℃ for 1min, and 45 cycles; the temperature is reduced to 4 ℃.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114317816A (en) * | 2021-10-28 | 2022-04-12 | 台州市药品检验研究院 | Kit for simultaneously detecting six common porcine viruses in compound cotyl raw materials and products thereof |
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| CN111955419A (en) * | 2020-08-31 | 2020-11-20 | 河南农业大学 | PDCoV and PEDV co-infected piglet morbidity model and establishment method and application thereof |
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| US20150299774A1 (en) * | 2012-06-27 | 2015-10-22 | Mobidiag Oy | Method for determining the presence of diarrhoea causing pathogens |
| CN104232801A (en) * | 2014-09-29 | 2014-12-24 | 四川农业大学 | Kit for detecting porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine rotavirus |
| CN105624330A (en) * | 2014-11-28 | 2016-06-01 | 北京亿森宝生物科技有限公司 | Taqman-MGB fluorescent quantitative PCR kit and method for detecting 12 common viruses and bacteria of pig at same time |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114317816A (en) * | 2021-10-28 | 2022-04-12 | 台州市药品检验研究院 | Kit for simultaneously detecting six common porcine viruses in compound cotyl raw materials and products thereof |
| CN114317816B (en) * | 2021-10-28 | 2023-09-01 | 台州市药品检验研究院 | Kit for simultaneously detecting six common viruses of pigs in compound koto raw materials and products thereof |
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