CN103937892B - The multiple PCR detection primer group of duck source various pathogens and test kit thereof - Google Patents
The multiple PCR detection primer group of duck source various pathogens and test kit thereof Download PDFInfo
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Abstract
The present invention relates to technical field of biological, specifically disclose multiple PCR detection primer group and the test kit thereof of duck source various pathogens.Described primer sets comprises 4 pairs of primers: RA rpoB-P1 and RA rpoB-P2; E.coli phoA-P1 and E.coli phoA-P2; Salm invA-P1 and Salm invA-P2; With Strep 16srRNA-P1 and Strep 16srRNA-P2.Multiplexed PCR amplification is carried out with above-mentioned four pairs of primers, can simultaneously quick, convenient, special, detect Riemerlla anatipestifer, intestinal bacteria, Salmonellas and suis four kinds of bacteriums delicately, can be used for Bacteria Identification, medical diagnosis on disease and epidemiology survey, there is wide market outlook and larger economic benefit.
Description
Technical field
The present invention relates to technical field of biological, be specifically related to multiple PCR detection primer group and the test kit thereof of duck source various pathogens.
Background technology
Riemerlla anatipestifer (
riemerellaAnatipestiferrA) be the pathogenic bacterium of the multiple bird infection morbidities such as duckling, poult and young goose, maximum to the duck harm in 1 ~ 8 week age, clinically in acute or chronic septicemia; pathology is based on fibrinous pericarditis, serohepatitis, airsacculitis, meningitis; the features such as caseous salpingitis, conjunctivitis, sacroiliitis appear in some cases, are commonly called as infectious serositis of duck.
Duck E．coli be pathogenic colon bacillus (
escherichia coli, E.coli) infect caused by, due to the difference of age of infected duck, resistibility and colibacillary virulence, route of infection, many different pathological changes and clinical symptom can be produced.Duckling to cause intestinal bacteria hepatitis and encephalitis for feature, at laying ducks there is escherichia coli Edeitis for characteristic.Intestinal bacteria are a kind of conditionality pathogenic bacterium, when various stress stimulation causes the immunologic function of duck to reduce, will infect.Therefore, the complication of riemerella anatipestifer disease is usually become clinically.
Duck streptococcicosis be by suis (
streptococcus,acute or the chronic infectious disease of the duck Strep) caused, it is acute death that duckling infects main manifestations, once morbidity, can cause mortality.The duck that grows up infects main manifestations for walking lamely and paralysing with kind duck, and mortality ratio is lower, comparatively large on the impact of production performance, causes larger financial loss.
Salmonella anatis disease is the general name of the disease caused by Salmonella typhimurium, Salmonella anatis and Salmonella enteritidis etc.The duck of various age in days can infect, and mainly occurs in duckling, can cause large quantities of death.This bacterioid can cause the food poisoning of people, significant in public health.
Riemerlla anatipestifer, intestinal bacteria, Salmonellas (
salmonella,salm) be the Main Pathogenic Bacteria causing duck fibrous pericarditis, serositis and serohepatitis, bring serious harm to countries in the world aviculture.Meanwhile, Riemerlla anatipestifer and suis polyinfection are also comparatively common.Main manifestations is serositis, easily causes mistaken diagnosis, if only by serositis treatment, though symptom can alleviate, mortality ratio declines to some extent, recurs again after drug withdrawal.
Above 4 kinds of bacterial infectious diseases all easily betide duckling, and easily polyinfection, all can cause serositis, its clinical symptom is similar with cuing open inspection pathology, is difficult to distinguish.At present traditional microbial culture, the method for biochemical identification and the PCR detection method etc. set up for single cause of disease are still mainly continued to use to the inspection of these pathogenic bacterium.The Isolation and ldentification of bacterium is the gold standard of diagnosis, but this method sense cycle is long, complex operation, and sensitivity is low and can not mass detection.
" foundation of Riemerellosis Anatipestifer Disease and colibacillosis differential diagnosis dual-PCR method and the application " of the research such as Qin Zonghua disclose a kind of can the dual-PCR method of quick and precisely differential diagnosis Riemerellosis Anatipestifer and colibacillosis, but the method also only can detect the bacteriosis of 2 kinds of ducks simultaneously, in order to cost-saving, improve detection efficiency, it is necessary for seeking a kind of method simultaneously detecting various bacteria disease.
Summary of the invention
Technical problem to be solved by this invention is the deficiency overcoming duck source pathogenic microbes detect technology in prior art, there is provided one group for the detection primer sets of duck source various pathogens, described pathogenic bacterium comprise Riemerlla anatipestifer, intestinal bacteria, suis and Salmonellas.
Another object of the present invention is to provide the test kit containing above-mentioned detection primer sets.
Another object of the present invention is to provide above-mentioned detection kit and is detecting the application in pest of duck various pathogens.
The object of the invention is to be achieved by the following technical programs:
The invention provides a kind of multiple PCR detection primer group for detecting 4 kinds of duck source pathogenic bacterium, described primer sets comprises 4 pairs of primers: RA rpoB-P1 and RA rpoB-P2; E.coli phoA-P1 and E.coli phoA-P2; Salm invA-P1 and Salm invA-P2; Strep 16srRNA-P1 and Strep 16srRNA-P2; Primer sequence is as shown in SEQ ID NO:1 ~ 8; Described 4 kinds of duck source pathogenic bacterium are Riemerlla anatipestifer, intestinal bacteria and Salmonellas and suis.
The nucleotide sequence of wherein said RA rpoB-P1 is as shown in SEQ ID NO:1, the nucleotide sequence of described RA rpoB-P2 is as shown in SEQ ID NO:2, the nucleotide sequence of described E.coli phoA-P1 is as shown in SEQ ID NO:3, the nucleotide sequence of described E.coli phoA-P2 is as shown in SEQ ID NO:4, the nucleotide sequence of described Strep16srRNA-P1 is as shown in SEQ ID NO:5, the nucleotide sequence of described Strep 16SrRNA-P2 is as shown in SEQ ID NO:6, the nucleotide sequence of described Salm invA-P1 is as shown in SEQ ID NO:7, the nucleotide sequence of described Salm invA-P2 is as shown in SEQ ID NO:8.
The present invention utilizes applicant to measure and is submitted to Riemerlla anatipestifer different serotypes on GenBank
rpoBthe sequence (GenBank accession numbers) of gene, with other bacteriums of delivering on GenBank as intestinal bacteria, suis, Salmonellas etc.
rpoBgene is compared, the zone design primer that Selective sequence difference is large, for the detection of Riemerlla anatipestifer.
The present invention selects colibacillary housekeeping gene
phoAgene as target gene, for the detection of E. coli isolated from ducks.To the different bacterium delivered
phoAgene order comparison redesigns primer.
The present invention selects streptococcic 16SrRNA gene as target gene, the title that 16SrRNA have " bacterial fossil ", this gene order almost can carry out the qualification on species level to all bacteriums, be in systematic bacteriology research the most frequently used, the most useful " molecular clock ".The present invention to compare redesign primer to delivering streptococcic 16SrRNA gene order.
The present invention selects Salmonellas
invAgene as target gene, for the detection of Salmonella anatis.To delivering Salmonellas
invAgene order is compared redesign primer.
Present invention also offers a kind of multiple PCR detection kit for detecting 4 kinds of duck source pathogenic bacterium, described test kit contains above-mentioned primer sets, and described 4 kinds of duck source pathogenic bacterium are Riemerlla anatipestifer, intestinal bacteria and Salmonellas and suis.
Preferably, in described multiple PCR detection kit, the upstream primer of each primer pair and the concentration of downstream primer are respectively 10 μMs, and described test kit is also containing 10 × PCR reaction buffer, archaeal dna polymerase and dNTP.Described 10 × PCR reaction buffer contains 100mM Tris-HCl (pH8.3), 500mM KCl and 15mM MgCl
2; Described archaeal dna polymerase concentration is 5units/ μ L, dNTP concentration is 2.5mM.
Preferably, the multi-PRC reaction system of described multiple PCR detection kit is: 10 × PCR reaction buffer 5 μ L, archaeal dna polymerase 0.3 μ L, dNTP 4 μ L, rpoB-P1 0.5 μ L, rpoB-P2 0.5 μ L, phoA-P1 0.5 μ L, phoA-P2 0.5 μ L, 16SrRNA-P1 0.5 μ L, 16SrRNA-P2 0.5 μ L, invA-P1 0.5 μ L, invA-P2 0.5 μ L, DNA profiling 1 μ L, with aseptic ultrapure water polishing to 25 μ L.
Preferably, the reaction conditions of the multiplex PCR of described multiple PCR detection kit is: 95 DEG C of denaturation 5min, 94 DEG C of 35s, 56 DEG C of 35s, and 72 DEG C of 45s run 35 circulations altogether, and last 72 DEG C extend 10min.
Present invention also offers above-mentioned multiple PCR detection kit and detect the application in pest of duck pathogenic bacterium.
Present invention also offers the using method of above-mentioned multiple PCR detection kit, comprise the following steps:
S1. testing sample DNA is extracted;
S2. mentioned reagent box is utilized to carry out multiplex PCR;
S3. the product after PCR is carried out agarose gel electrophoresis, carry out result judgement, described result is judged to be: if there is 139bp, 256bp, 327bp, and 482bp band, then show that sample contains Riemerlla anatipestifer and intestinal bacteria and suis and Salmonellas; If there is 139bp, 256bp and 327bp, then show that sample contains Riemerlla anatipestifer and intestinal bacteria and suis; If there is 139bp, 256bp and 482bp band, then show that sample contains Riemerlla anatipestifer and intestinal bacteria and Salmonellas; If there is 256bp, 327bp and 482bp band, then show that sample contains intestinal bacteria and suis and Salmonellas; If there is 139bp, 327bp and 482bp band, then show that sample contains Riemerlla anatipestifer and suis and Salmonellas; If there is 139bp and 256bp band, then show that sample contains Riemerlla anatipestifer and intestinal bacteria; If there is 139bp, 327bp band, then show that sample contains Riemerlla anatipestifer and suis; If there is 139bp and 482bp band, then show that sample contains Riemerlla anatipestifer and Salmonellas; If there is 256bp and 327bp band, then show that sample contains and intestinal bacteria and suis; If there is 256bp and 482bp band, then show sample large intestine bar and Salmonellas; If there is 327bp and 482bp band, then show that sample contains suis and Salmonellas; If there is 139bp band, then show that sample contains Riemerlla anatipestifer; If there is 256bp band, then show that sample contains intestinal bacteria; If there is 327bp band, then show that sample contains suis; If there is 482bp band, then show that sample contains Salmonellas.
Compared with prior art, beneficial effect of the present invention:
The invention provides a kind of multiple PCR detection primer group and the multiple PCR detection kit containing described primer sets, described primer sets or test kit are for detecting duck source various pathogens, not only greatly shorten detection time, can also detect sample by batch, can simultaneously quick, convenient, special, detect Riemerlla anatipestifer, intestinal bacteria, Salmonellas and suis four kinds of bacteriums delicately.
Present invention also offers above-mentioned primer sets or the application of test kit in detection pest of duck pathogenic bacterium.Described primer sets or test kit quick, easy, be quick on the draw, can be used for Bacteria Identification, medical diagnosis on disease and epidemiology survey, there is wide market outlook and larger economic benefit.
Accompanying drawing explanation
Fig. 1 multiplex PCR specific detection; M:Takara DL500 marker; Swimming lane a: Riemerlla anatipestifer; Swimming lane b: intestinal bacteria; Swimming lane c: suis; Swimming lane d: Salmonellas; Swimming lane e: Riemerlla anatipestifer+intestinal bacteria; Swimming lane f: Riemerlla anatipestifer+suis; Swimming lane g: Riemerlla anatipestifer+Salmonellas; Swimming lane h: intestinal bacteria+suis; Swimming lane i: intestinal bacteria+Salmonellas; Swimming lane j: suis+Salmonellas; Swimming lane k: Riemerlla anatipestifer+intestinal bacteria+suis; Swimming lane l: intestinal bacteria+suis+Salmonellas; Swimming lane m: Riemerlla anatipestifer+intestinal bacteria+Salmonellas; Swimming lane n: Riemerlla anatipestifer+suis+Salmonellas; Swimming lane o: Riemerlla anatipestifer+intestinal bacteria+suis+Salmonellas; Swimming lane p: negative control (deionized water);
Fig. 2 multiplex PCR susceptibility detects; M:Takara DL500 marker; Swimming lane a:4 × 10
4copy; Swimming lane b:4 × 10
3copy; Swimming lane c:4 × 10
2copy; Swimming lane d:4 × 10
1copy; Swimming lane e: negative control (deionized water);
Fig. 3 Riemerlla anatipestifer, intestinal bacteria, suis and Salmonellas laboratory infection clinical sample detect; Swimming lane M:Takara DL2000marker; Swimming lane a: the hepatic tissue of Riemerlla anatipestifer (serotype 1 type) infected duck; Swimming lane b: the cerebral tissue of Riemerlla anatipestifer (serotype 1 type) infected duck; The hepatic tissue of swimming lane c: large intestine bar ATCC8099 strain infected duck; The cerebral tissue of swimming lane d: intestinal bacteria ATCC8099 strain infected duck; The hepatic tissue of swimming lane e: suis CVCC556 strain infected duck; The cerebral tissue of swimming lane f: suis CVCC556 strain infected duck; Swimming lane g: the hepatic tissue of Salmonella Typhimurium Infection duck; Swimming lane h: the cerebral tissue of Salmonella Typhimurium Infection duck; Swimming lane i: Riemerlla anatipestifer+intestinal bacteria+suis+Salmonellas; Swimming lane j: negative control (deionized water).
Embodiment
Below in conjunction with Figure of description and specific embodiment, set forth the present invention further.These embodiments are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in lower routine embodiment, the usually conveniently conditioned disjunction condition of advising according to manufacturer.Unless otherwise defined, the same meaning that all specialties used in literary composition and scientific words and those skilled in the art are familiar with.
embodiment 1 design of primers and primer specificity detect
The present embodiment utilizes applicant to measure and is submitted to Riemerlla anatipestifer different serotypes on GenBank
rpoBthe sequence (GenBank accession numbers) of gene, with other bacteriums of delivering on GenBank as intestinal bacteria, suis, Salmonellas etc.
rpoBgene is compared, the zone design primer that Selective sequence difference is large, and for the detection of Riemerlla anatipestifer, described primer pair is RA rpoB-P1(SEQ ID NO:1) and RArpoB-P2(SEQ ID NO:2).
The present embodiment selects colibacillary housekeeping gene
phoAgene as target gene, for the detection of E. coli isolated from ducks.To the different bacterium delivered
phoAgene order comparison redesigns primer, and described primer pair is E.coli phoA-P1(SEQ ID NO:3) and E.coli phoA-P2(SEQ ID NO:4).
The present embodiment selects streptococcic 16SrRNA gene as target gene, redesign primer to delivering streptococcic 16SrRNA gene order, described primer pair is Strep16srRNA-P1(SEQ ID NO:5) and Strep16srRNA-P2(SEQ ID NO:6).
The present embodiment selects Salmonellas
invAgene as target gene, for the detection of Salmonella anatis.To delivering Salmonellas
invAgene order redesigns primer, and described primer pair is Salm invA-P1(SEQ ID NO:7) and Salm invA-P2(SEQ ID NO:8).
Above-mentioned 4 pairs of primer sequences are as follows:
SEQ ID NO：1：5’-TGCCCAAGCGAATGTGGAGC-3’；
SEQ ID NO：2：5’-ACCGGAAATCTGGTTTGGCG-3’；
SEQ ID NO：3：5’-CGATAAGCCCGCAGTCACCT-3’；
SEQ ID NO：4：5’-GACCAGCGTGTTACCCTCCT-3’；
SEQ ID NO：5：5’-TACCAGAAAGGGACGGCTAA-3’；
SEQ ID NO：6：5’-CGTTTACGGCGGCGTGGACTACC-3’；
SEQ ID NO：7：5’-TGGGTAACGCATGAAGAGGG-3’；
SEQ ID NO：8：5’-GGGTCAAGGCTGAGGAAGGT-3’。
Carry out PCR according to designed 4 pairs of Specific PCR primers to 26 strain common pathogens and detect negative, positive findings, wherein 10 strain Riemerlla anatipestifers all detect rpoB gene, and all non-Riemerlla anatipestifers are showed no above-mentioned specific amplification band; 5 strain intestinal bacteria all detect phoA gene, and all non-intestinal bacteria are showed no above-mentioned specific amplification band; 2 strain suis all detect 16SrRNA gene, and all non-suis are showed no above-mentioned specific amplification band; 5 strain Salmonellass all detect invA gene, and all nonsalmonella have no above-mentioned specific amplification band.Namely show that 4 pairs of primers have good specificity.
the foundation of embodiment 2 duck source various pathogens multiple PCR detection kit
By following composition preparation duck source pathogenic bacteria multiple PCR detection kit:
Containing 10 × PCR reaction buffer (100mM Tris-HCl (pH8.3), 500mM KCl, 15mM MgCl
2), archaeal dna polymerase (5units/ μ L), dNTP(2.5mM), the sequence of 4 pairs of primers (10uM) is as follows:
SEQ ID NO：1：5’-TGCCCAAGCGAATGTGGAGC-3’；
SEQ ID NO：2：5’-ACCGGAAATCTGGTTTGGCG-3’；
SEQ ID NO：3：5’-CGATAAGCCCGCAGTCACCT-3’；
SEQ ID NO：4：5’-GACCAGCGTGTTACCCTCCT-3’；
SEQ ID NO：5：5’-TACCAGAAAGGGACGGCTAA-3’；
SEQ ID NO：6：5’-CGTTTACGGCGGCGTGGACTACC-3’；
SEQ ID NO：7：5’-TGGGTAACGCATGAAGAGGG-3’；
SEQ ID NO：8：5’-GGGTCAAGGCTGAGGAAGGT-3’。
The present invention is optimized above-mentioned condition, finally determine that the reaction system of this test kit is: 10 × PCR reaction buffer 5 μ L, archaeal dna polymerase 0.3 μ L, dNTP 4 μ L, rpoB-P1 0.5 μ L, rpoB-P2 0.5 μ L, phoA-P1 0.5 μ L, phoA-P2 0.5 μ L, 16SrRNA-P1 0.5 μ L, 16SrRNA-P2 0.5 μ L, invA-P1 0.5 μ L, invA-P2 0.5 μ L, DNA profiling 1 μ L, with aseptic ultrapure water polishing to 25 μ L.
Above-mentioned DNA profiling is made up of the DNA of these 4 kinds of bacteriums of intestinal bacteria, Salmonellas, suis and Riemerlla anatipestifer, and the leaching process of above-mentioned DNA of bacteria is: the single intestinal bacteria of picking and Salmonellas bacterium colony respectively in 3mL LB broth culture in 37 DEG C of 200 rpm shaking table overnight incubation; The single suis of picking and Riemerlla anatipestifer bacterium colony to add in the TSB substratum of 5% serum overnight incubation in 37 DEG C of 200 rpm shaking table respectively at 3mL.The bacterium liquid of the above-mentioned 4 kinds of bacteriums of gained, in the centrifugal 5min of 12000 rpm, abandons supernatant, and after resuspended with 200 μ L 1 × TE solution, boil 10 min, ice bath 5 min, the centrifugal 5min of 12000 rpm, gets supernatant ,-20 DEG C of preservations.
The reaction conditions of above-mentioned multiplex PCR is: 95 DEG C of denaturation 5 min, and at 94 DEG C of 35 s, 56 DEG C of 35 s, 72 DEG C of 45 s runs 35 circulations altogether, and last 72 DEG C extend 10 min.
The qualification of above-mentioned multiplexed PCR amplification product: get after 5 μ L PCR primer mix with 6 × loading Buffer and be added in the sepharose of 2.5%, deposition condition is 120V, 30min; With Takara DL 500 marker for standard reference, under ultraviolet imager, observe detected result.Detect show Riemerlla anatipestifer rpoB, intestinal bacteria phoA, the length of suis 16SrRNA and these 4 kinds of target genes of Salmonellas invA is respectively 139bp, 256bp, 327bp, 482bp.
the specificity of multiple PCR detection kit described in embodiment 3
Prepare the 18 strain bacterium such as 107 cfu/mL Riemerlla anatipestifers (serotype 1 type), intestinal bacteria (ATCC8099), suis (CVCC556), Salmonella typhimurium, avian pasteurella multocida, streptococcus aureus, Pseudomonas aeruginosa and mycoplasma respectively, extract the DNA profiling of 18 strain bacterium with water-boiling method.The single bacterial strain DNA, between two the hybrid bacterial strain DNA that Riemerlla anatipestifer, intestinal bacteria, suis and Salmonellas 4 strain bacterium are extracted, three or three hybrid bacterial strain DNA and four strain hybrid bacterial strain DNA are template, the single bacterial strain DNA that avian pasteurella multocida, streptococcus aureus, Pseudomonas aeruginosa and mycoplasma 4 strain bacterium are extracted is template, PCR reaction is carried out, to determine the specificity of multi-PRC reaction under the PCR condition optimized described in embodiment 2.Observe after electrophoresis is carried out to PCR reaction product, result as shown in Figure 1, Fig. 1 result shows except Riemerlla anatipestifer of the present invention, intestinal bacteria, suis and Salmonellas, other bacterium bands all do not detect, and illustrate that the present invention can detect Riemerlla anatipestifer, intestinal bacteria, suis and Salmonellas specifically.
the susceptibility of multiple PCR detection kit described in embodiment 4
PMD18-T Vector test kit single PCR primer is used to carry out glue recovery, glue reclaims product and is connected with 16 DEG C, 18-T carrier and spends the night, then use DH5 α competent cell to transform, at 200 rpm 37 DEG C, cultivate 1 h, draw 100 μ L and be coated on SOC substratum and spend the night.Picking white colony, in 5mL LB meat soup overnight incubation, uses omega test kit to extract plasmid and checks order, and detect plasmid concentration.Carried out the estimation of plasmid copy number by formula, the copy number of unified often kind of plasmid, and prepare the hybrid template of four bacterium.The hybrid template ddH obtained
20 carries out 10 times of dilutions.In PCR reaction, add 1 μ L hybrid template respectively, make often kind of plasmid template final concentration under each extent of dilution be 4 × 10
4copy, 4 × 10
3copy, 4 × 10
2copy, 4 × 10
1copy.Increase with the PCR condition optimized described in embodiment 2, thus determine the susceptibility of multiplex PCR, as shown in Figure 2, Fig. 2 result is that Riemerlla anatipestifer, intestinal bacteria and Salmonellas are 4 × 10 to result
2copy, suis is 4 × 10
3copy.
described in embodiment 5, multiple PCR detection kit is to the detection of laboratory sample
The 80 strain Riemerlla anatipestifers using the multiple PCR detection kit of embodiment 2 foundation to preserve laboratory, 96 strain intestinal bacteria, 12 strain suis and 142 strain Salmonellas clinical separation strains detect.
(1) DNA of bacteria Template preparation
The single bacterium colony of Riemerlla anatipestifer, intestinal bacteria, suis and Salmonellas is inoculated into TSB+5% serum or LB liquid nutrient medium respectively, and 37 DEG C are shaken bacterium.Intestinal bacteria and Salmonellas shake bacterium 4 h, and Riemerlla anatipestifer and suis shake bacterium 8 h.Respectively get 3mL bacterium liquid and extract DNA with water-boiling method in EP pipe ,-20 DEG C of preservations.And establish known Riemerlla anatipestifer, intestinal bacteria, suis and Salmonellas 4 kinds of bacterium mixed bacteria liquid water-boiling methods to extract DNA, as positive control; Replace DNA profiling as negative control using deionized water in addition.
(2) PCR reaction system
The multiplex PCR system after optimizing is adopted to detect: 10 × Buffer 5 μ L, archaeal dna polymerase 0.3 μ L, dNTP 4 μ L, rpoB-P1 0.5 μ L, rpoB-P2 0.5 μ L, phoA-P1 0.5 μ L, phoA-P2 0.5 μ L, 16SrRNA-P1 0.5 μ L, 16SrRNA-P2 0.5 μ L, invA-P1 0.5 μ L, invA-P2 0.5 μ L, DNA profiling 1 μ L, with aseptic ultrapure water polishing to 25 μ L.
(3) multi-PRC reaction condition: 95 DEG C of denaturation 5 min, at 94 DEG C of 35 s, 56 DEG C of 35 s, 72 DEG C of 45 s runs 35 circulations altogether, and last 72 DEG C extend 10 min.
(4) multiplex PCR detected result: corresponding band has appearred in the positive position of detected result display Riemerlla anatipestifer, intestinal bacteria, suis and Salmonellas 4 kinds of bacteriums, namely presents specific reaction.Namely show that the present invention can carry out specific detection to the clinical separation strain of above-mentioned 4 kinds of bacteriums.
described in embodiment 6, multiple PCR detection kit is to the detection of clinical sample
8 increment product have infected Riemerlla anatipestifer, intestinal bacteria, the cerebral tissue of duck of suis or Salmonellas and liver organization respectively from laboratory.The liver of 24 parts of ducks that die of illness, cerebral tissue all come that idiopathy duck field collects, and to suspect the being duck that dies of illness of bacteriological infection.
(1) preparation of DNA profiling
Liver, the cerebral tissue of a small amount of duck of aseptic technique transfering loop picking, be inoculated in TSB+5% sera liquid substratum, 37 DEG C are shaken bacterium overnight incubation.Get 3mL bacterium liquid and extract DNA with water-boiling method in EP pipe ,-20 DEG C of preservations.And establish known Riemerlla anatipestifer, intestinal bacteria, suis and Salmonellas 4 kinds of bacterium mixed bacteria liquid water-boiling methods to extract DNA, as positive control; Replace DNA profiling as negative control using deionized water in addition.
(2) PCR reaction system
The multiplex PCR system after optimizing is adopted to detect: 10 × Buffer 5 μ L, archaeal dna polymerase 0.3 μ L, dNTP 4 μ L, rpoB-P1 0.5 μ L, rpoB-P2 0.5 μ L, phoA-P1 0.5 μ L, pho A-P2 0.5 μ L, 16SrRNA-P10.5 μ L, 16SrRNA-P2 0.5 μ L, invA-P1 0.5 μ L, invA-P2 0.5 μ L, DNA profiling 1 μ L, with aseptic ultrapure water polishing to 25 μ L.
(3) multi-PRC reaction condition: 95 DEG C of denaturation 5 min, at 94 DEG C of 35 s, 56 DEG C of 35 s, 72 DEG C of 45 s runs 35 circulations altogether, and last 72 DEG C extend 10 min.
(4) multiplex PCR detected result
The results are shown in Figure 3 after carrying out multiplex PCR detection with test kit described in embodiment 2,8 parts are infected Riemerlla anatipestifer, intestinal bacteria, the liver of suis or Salmonellas duck and brain tissue sample respectively from laboratory, and detecting with multiplex PCR is all the positive.24 ducks (detecting liver and cerebral tissue), there are 2 ducks to detect Riemerlla anatipestifer and intestinal bacteria simultaneously, there are 3 ducks to detect intestinal bacteria and suis simultaneously, 2 ducks are had only to detect Riemerlla anatipestifer, 4 ducks are had to detect intestinal bacteria, there are 11 ducks only to detect suis, have 2 ducks only to detect Salmonellas.The result of bacteria distribution and qualification also demonstrate that the exactness of multiplex PCR result.
SEQUENCE LISTING
<110> Agricultural University Of South China
The multiple PCR detection primer group of <120> duck source various pathogens and test kit thereof
<130>
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> RA rpoB-P1
<400> 1
tgcccaagcg aatgtggagc 20
<210> 2
<211> 20
<212> DNA
<213> RA rpoB-P2
<400> 2
accggaaatc tggtttggcg 20
<210> 3
<211> 20
<212> DNA
<213> E.coli phoA-P1
<400> 3
cgataagccc gcagtcacct 20
<210> 4
<211> 20
<212> DNA
<213> E.coli phoA-P2
<400> 4
gaccagcgtg ttaccctcct 20
<210> 5
<211> 20
<212> DNA
<213> Strep16srRNA-P1
<400> 5
taccagaaag ggacggctaa 20
<210> 6
<211> 23
<212> DNA
<213> Strep16srRNA-P2
<400> 6
cgtttacggc ggcgtggact acc 23
<210> 7
<211> 20
<212> DNA
<213> Salm invA-P1
<400> 7
tgggtaacgc atgaagaggg 20
<210> 8
<211> 20
<212> DNA
<213> Salm invA-P2
<400> 8
gggtcaaggc tgaggaaggt 20
Claims (4)
1. for detecting a multiple PCR detection primer group for 4 kinds of duck source pathogenic bacterium, it is characterized in that, described primer sets comprises 4 pairs of primers: RA rpoB-P1 and RA rpoB-P2; E.coli phoA-P1 and E.coli phoA-P2; Salm invA-P1 and Salm invA-P2; Strep 16srRNA-P1 and Strep 16srRNA-P2; Primer sequence is as shown in SEQ ID NO:1 ~ 8; Described 4 kinds of duck source pathogenic bacterium are Riemerlla anatipestifer, intestinal bacteria and Salmonellas and suis.
2. for detecting a multiple PCR detection kit for 4 kinds of duck source pathogenic bacterium, it is characterized in that, described test kit contains primer sets described in claim 1, and described 4 kinds of duck source pathogenic bacterium are Riemerlla anatipestifer, intestinal bacteria and Salmonellas and suis.
3. multiple PCR detection kit according to claim 2, it is characterized in that, in described test kit, the upstream primer of each primer pair and the concentration of downstream primer are respectively 10 μMs, and described test kit is also containing 10 × PCR reaction buffer, archaeal dna polymerase and dNTP.
4. multiple PCR detection kit according to claim 2, it is characterized in that, the reaction conditions of the multiplex PCR of described test kit is: 95 DEG C of denaturation 5min, 94 DEG C of 35s, 56 DEG C of 35s, and 72 DEG C of 45s run 35 circulations altogether, and last 72 DEG C extend 10min.
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| CN105154432B (en) * | 2015-09-17 | 2018-04-06 | 北京工业大学 | A kind of method and its multiple PCR primer group for being used to expand four genes of the pathogen of Botrytis cinerea |
| CN106947834B (en) * | 2017-04-06 | 2020-08-21 | 江苏农牧科技职业学院 | Multiplex PCR method for detecting six duck susceptibility viruses |
| CN107254534A (en) * | 2017-07-05 | 2017-10-17 | 中国农业科学院兰州兽医研究所 | A kind of kit of TaqMan probe fluorescence quantitative PCR detection mycoplasma anatis |
| CN110117671A (en) * | 2019-06-25 | 2019-08-13 | 江苏省家禽科学研究所 | Riemerellosis Anatipestifer Specific PCR primers are to, PCR detection method and kit |
| CN110904251B (en) * | 2019-11-28 | 2023-01-13 | 温氏食品集团股份有限公司 | Primer group and kit for detecting duck mycoplasma |
| CN111534619A (en) * | 2020-05-11 | 2020-08-14 | 贵州省畜牧兽医研究所 | Multiple PCR detection method and detection kit for chicken-derived pathogenic bacteria |
| CN112195258B (en) * | 2020-10-26 | 2023-10-13 | 贵州省畜牧兽医研究所 | Multiplex PCR detection kit for multiple pathogenic bacteria of waterfowl and application thereof |
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