CN108559784B - Triple PCR detection primer, probe, kit and detection method for simultaneously detecting three pathogenic bacteria - Google Patents
Triple PCR detection primer, probe, kit and detection method for simultaneously detecting three pathogenic bacteria Download PDFInfo
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Abstract
The invention discloses a primer, a probe, a kit and a detection method for triple PCR detection for simultaneously detecting three pathogenic bacteria. The invention provides specific primers aiming at the genes of Plo, ICEMh1 and pspC, and the DNA sequence of the primers is shown as SEQ ID NO. 1-SEQ ID NO. 6; meanwhile, a probe is provided, and the DNA sequence of the probe is shown as SEQ ID NO. 7-SEQ ID NO. 9. Based on the primers and the probes, the invention optimizes a real-time fluorescent PCR detection system and reaction program conditions, successfully establishes a high-flux triple real-time fluorescent quantitative PCR detection method of the cryptobacter pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae TaqMan probes, has simple and safe operation, higher sensitivity, specificity and stability, can well meet the rapid detection and identification of the cryptobacter pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae in the breeding industry, can analyze the carrying conditions of three virulence genes of three bacteria, and can be used for epidemiological traceability and virulence strength analysis.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a primer, a probe, a kit and a detection method for triple PCR detection for simultaneously detecting three pathogenic bacteria.
Background
Cryptobacterium pyogenes (Arcanobacterium pyogenes) is a gram-positive short corynebacterium, and is mainly responsible for pyogenic infections of various organs of animals such as cattle, sheep, pigs and humans, such as pneumonia, arthritis, endocarditis, mastitis, abscess, osteomyelitis and metritis. Mannheimia haemolytica is a gram-negative coccus, one of the most important respiratory pathogens in ruminants, and causes epidemic pneumonia in ruminants and wildlife. Streptococcus pneumoniae is a gram-positive coccus, can cause a plurality of important pathogens of infection, is colonized in the upper respiratory tract of an organism, and causes a series of fatal infections such as pneumonia, otitis media, bacteremia and the like worldwide. The cryptococcus pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae from different sources have larger virulence difference, and the accurate and rapid detection of the cryptococcus pyogenes, the mannheimia haemolytica, the streptococcus pneumoniae and virulence genes thereof has important significance for preventing the cryptococcus pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae and also provides important technical support for the traceability and risk assessment of food-borne cryptococcus pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae.
The PLO gene, the ICEMh1 gene and the pspC gene are respectively important genes of cryptococcus pyogenes, mannheimia haemolytica and streptococcus pneumoniae, and the hemolysin is exotoxin and can dissolve a plurality of animal erythrocytes and immune cells to cause death of animals. ICEMh1 is a mobile genetic element that normally integrates into the host's chromosomal DNA at a specific site and passively propagates during chromosomal replication and cell division. ICEMh1 is also an antibiotic resistance gene. The pspC is a surface protein that stimulates the synthesis of IL-8 by lung epithelial cells and is involved in the activation and chemotaxis of host immune cells. The pspC mediates the adhesion of glycopeptides and sialic acid residues of epithelial cells, and promotes the adhesion and penetration of bacteria by recognizing and polymerizing Ig receptors and secretory IgA binding.
According to the traditional detection method for bacteria separation and identification, a suspicious bacterial colony is subjected to biochemical identification after two times of enrichment culture, the detection period is as long as 6-7 d, and the requirement of large-batch and rapid detection cannot be met; the situation of the major virulence genes it carries is also unknown. The detection of the cryptobacter pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae is carried out by adopting the traditional bacteria biochemical identification and single PCR (fluorescent PCR) method for a long time in the field, and the detection is time-consuming, complicated to operate and low in sensitivity. Moreover, the virulence gene conditions carried by the cryptococcus pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae cannot be analyzed, and the virulence strength of the cryptococcus pyogenes, the mannheimia haemolytica and the epidemiological traceability of the cryptococcus pyogenes cannot be carried out by the method. The requirements for risk monitoring of cryptobacter pyogenes, mannheimia haemolytica and streptococcus pneumoniae cannot be met.
Disclosure of Invention
The invention aims to solve the technical problem of providing a primer, a probe, a kit and a detection method for triple PCR detection for simultaneously detecting three pathogenic bacteria based on a Taqman probe, aiming at the defects of detection technologies of cryptococcus pyogenes, mannheimia haemolytica and streptococcus pneumoniae. The Taqman probe has the advantages of high resolution and strong specificity, a high-throughput detection method with strong specificity, high sensitivity and good stability for the cryptococcus pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae is established, and the conditions that the cryptococcus pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae carry three specific genes, namely plo gene and ICEMh1 gene pspC gene, can be detected. Facilitates the strong and weak and tracing analysis of the toxicity of the strains of the cryptobacter pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae.
The invention is realized by the following technical scheme:
aiming at the cryptococcus pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae, the invention selects three specific virulence genes through the pertinence analysis of the carried virulence genes of the cryptococcus pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae: plo gene, ICEMh1 gene, pspC gene, and then aligned using DNASAR according to the sequences of Cryptococcus pyogenes, Mannheim hemolyticus, and Streptococcus pneumoniae plo, ICEMh1, and pspC genes published on GenBank, the sequences were analyzed and primers and probes were designed using primer express 3.0 in the conserved regions thereof. A method for detecting the cryptobacter pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae by triple real-time fluorescent quantitative PCR is established.
First, the present invention provides a primer for real-time fluorescent PCR detection of the crypthecium pyogenes plo gene, the mannheim haemolyticus ICEMh1 gene, and the streptococcus pneumoniae pspC gene, wherein:
DNA sequences of an upstream primer and a downstream primer of Plo gene of the cryptobacter pyogenes are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2;
DNA sequences of upstream and downstream primers of ICEMh1 gene of Mannheimia haemolytica are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4;
the DNA sequences of the upstream and downstream primers of the pspC gene of Streptococcus pneumoniae are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.
Secondly, the invention provides a TaqMan triple real-time fluorescence PCR detection probe for cryptobacter pyogenes, mannheimia haemolytica and streptococcus pneumoniae, wherein:
the DNA sequence of the Plo gene probe of the cryptococcus pyogenes is shown in SEQ ID NO.7, the 5 'end of the Plo gene probe is marked with a Cy5 fluorescent reporter group, and the 3' end of the Plo gene probe is marked with BHQ 1;
the DNA sequence of the ICEMh1 gene probe of Mannheimia haemolytica is shown in SEQ ID NO.8, the 5 'end of the ICEMh1 gene probe is marked with a VIC fluorescent reporter group, and the 3' end of the ICEMh1 gene probe is marked with BHQ 1;
the DNA sequence of the pspC gene probe of Streptococcus pneumoniae is shown in SEQ ID NO.9, and the 5 'end of the pspC gene probe is labeled with FAM fluorescent reporter group, and the 3' end is labeled with BHQ 1.
The invention also provides a TaqMan triple real-time fluorescence PCR detection kit for the cryptobacter pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae, wherein the total volume of a reaction system in the kit is 25 mu L, and the kit comprises:
Premix Ex TaqTM(2×)12.5μL；
plo primer, probe (10. mu.M), 0.5. mu.L each;
0.5. mu.L each of primer ICEMh1 and probe (10. mu.M);
0.5. mu.L each of the pspC primer and probe (10. mu.M);
ROX 0.3μL；
5.7 mu L of deionized water; the DNA template was 2. mu.L;
wherein, the DNA sequences of the Plo gene upstream primer, the Plo gene downstream primer and the Plo gene probe are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 7;
the DNA sequences of the IICEMh1 gene upstream primer, the IICEMh1 gene downstream primer and the IICEMh1 gene probe are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 8;
the DNA sequences of the pspC gene upstream primer, the pspC gene downstream primer and the pspC gene probe are respectively shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.9
Finally, the invention also provides a TaqMan triple real-time fluorescence quantitative PCR detection method for the cryptobacter pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae, which comprises the following steps:
s1, sucking 1mL of overnight culture solution at 37 ℃, centrifuging in a 2mL centrifuge tube to remove the culture medium, rinsing with 1mL of deionized water, centrifuging at 12000r/min for 2min to remove the supernatant, and repeating for 2 times. DNA was extracted for use according to kit instructions.
S2, carrying out real-time fluorescence PCR detection by adopting the primer, the probe and the detection method designed by the invention.
S3, carrying out quantitative detection on the cryptobacter pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae by adopting the method established by the invention.
Preferably, the specific method for extracting DNA by S1 is as follows: after the bacterial strain is subjected to enrichment culture for 24 hours by using brain heart extract liquid, 1mL of bacterial liquid is taken for 12000r/min, the supernatant is centrifuged, and DNA is extracted by adopting a commercialized bacterial genome DNA extraction kit to serve as a reaction template.
Preferably, the triple real-time fluorescent PCR detection reaction system in S2 is 25 μ L: plo, ICEMh1, and pspC primer probes (10. mu.M) were 0.5. mu.L, ROX 0.3. mu.L, and DI water 5.7. mu.L of DNA template 2. mu.L, respectively. Reaction conditions are as follows: stage 1: pre-denaturation at 95 ℃ for 30 s; stage 2: collecting fluorescence signals from three channels of FAM, VIC and Cy5 at 95 ℃ for 5s, 60 ℃ for 34s and 40 cycles; the amplification reaction was performed on an ABI7500FAST fluorescent quantitative PCR instrument.
Preferably, the reaction procedure of the triple real-time fluorescent PCR detection in S2 is: stage 1: pre-denaturation at 95 ℃ for 30 s; stage 2: collecting fluorescence signals at 95 ℃ for 5s, 60 ℃ for 34s, 40 cycles and three channels of Cy5, VIC and FAM; the qualitative results of the cryptobacter pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae are as follows: and if the Ct value of the gene to be detected is less than 36, the gene to be detected is judged to be positive, if the Ct value is less than 36 and less than 40, the gene to be detected is judged to be suspicious, the template quantity can be increased for repeated experiments, if a typical amplification curve exists, the gene to be detected is judged to be positive, otherwise, the gene to be detected is negative, and no Ct value or Ct value equal to 40 is negative.
Preferably, the method for quantitatively detecting the cryptobacter pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae in S3 comprises the following steps:
the plo, ICEMh1 and pspC gene fragment sequences amplified by the strain are adopted to be cloned in puc57(EcoRV, AmpR resistance) and construct a.p-m.h1-s.p plasmid, and 1.25 multiplied by 10 is adopted6、1.25×105、1.25×104、1.25×103250, 50, and 10 copies/. mu.L 7 plasmid DNA dilutions and performing triple real-time fluorescent quantitative PCR amplification as S2 with Ct value on the Y-axis and log on the X-axis10(CFU) respectively drawing plo, ICEMh1 and pspC gene standard curves, and substituting the standard curves into the Ct value obtained by amplification of the sample to be detected according to the formulas of the three standard curves to calculate the contents of cryptobacter pyogenes, mannheimia haemolytica and streptococcus pneumoniae in the sample.
Compared with the prior art, the invention has the following beneficial technical effects:
the invention relates to a triple real-time fluorescent PCR (polymerase chain reaction) rapid detection method for cryptobacter pyogenes, mannheimia haemolytica and streptococcus pneumoniae, which is established by using a TaqMan probe technology, wherein 5' is marked by adopting different fluorescent reporter groups, signals are not interfered with each other, the combination rigor of the probe is improved, the difference of one base can be identified at most, and the detection resolution is increased. Meanwhile, the quenching group of the TaqMan probe is a non-fluorescent quenching group, so that fluorescence is not generated, the intensity of a fluorescence background signal of real-time fluorescence PCR reaction is reduced, and the sensitivity of the TaqMan probe is further improved. The same sample adopts three pairs of primers to respectively amplify different target gene fragments, and three genes of plo, ICEMh1 and pspC are detected simultaneously, so that the efficiency is improved, the time is saved, and the detection cost is reduced. Not only can rapidly detect the cryptococcus pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae, but also can detect the carrying conditions of the genes plo, ICEMh1 and pspC of the cryptococcus pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae, provides a reference basis for analyzing the virulence of the cryptococcus pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae, and also provides convenience for the cluster typing of the cryptococcus pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae and the traceability of epidemiology, the kit has important significance for controlling spread of diseases caused by the cryptococcus pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae and treatment of the cryptococcus pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae, and is suitable for rapid detection of the carrying conditions of the virulence genes of the cryptococcus pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae and epidemiological investigation of a large number of samples.
The Taqman probe used by the invention is used for carrying out fluorescence quantitative PCR detection, and the advantages are as follows:
1, the operation is simple and convenient, the qualitative judgment can be visually carried out through an amplification curve without subsequent steps such as electrophoresis and the like, the whole PCR process is monitored through the accumulation of fluorescence signals, then the unknown template is qualitatively or quantitatively analyzed through the amplification curve or Ct value, and the rapid detection and identification of the cryptobacter pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae and the analysis of the carrying conditions of three virulence genes can be well met;
2. rapid, high throughput: the invention can detect 20 copies of standard substance positive plasmid with the lower limit of quantification, the whole amplification process is within 45 minutes, three virulence genes can be detected at one time, the detection cost is saved, and high-flux detection can be carried out;
3. the standard curves of the three virulence genes obtained by the invention can realize the quantitative detection of the cryptobacter pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae.
Drawings
FIG. 1 is a diagram showing the specific results of TaqMan triple real-time fluorescent PCR detection.
FIG. 2 is a graph showing the sensitivity results of TaqMan triple real-time fluorescent PCR detection.
FIG. 3 is a standard curve for TaqMan triple real-time fluorescent PCR detection.
Detailed Description
The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention. Unless otherwise specified, the biomaterials, reagent raw materials used in the following examples are conventionally commercially available or commercially available biomaterials and reagent raw materials, and unless otherwise specified, the methods and apparatuses used in the following examples are those conventionally used in the art.
1. Establishing a triple real-time fluorescent PCR detection system for cryptobacter pyogenes, mannheimia haemolytica and streptococcus pneumoniae
1.1 design of primers and probes
Three important virulence genes were selected by targeted analysis of the virulence-carrying genes of cryptobacter pyogenes, mannheimia haemolytica and streptococcus pneumoniae: plo, ICEMh1 and pspC, then aligned with DNAStar according to the published sequences of the Cryptococcus pyogenes plo gene, the Mannheim haemolyticus ICEMh1 gene and the Streptococcus pneumoniae pspC gene on GenBank, the sequences were analyzed and primers and probes were designed in their conserved regions using PrimerExpress 3.0, the sequences of the primers were:
SEQ ID NO.1：plo-F：5’-ttgacggacggcttgtca-3’。
SEQ ID NO.2：plo-R：5’-gcgccgacttaaggtcaact-3’。
SEQ ID NO.3：ICEMh-F：5’-ccacgccaaccgatgaa-3’。
SEQ ID NO.4：ICEMh-R：5’-agagtgccggcatagcctaa-3’。
SEQ ID NO.5：pspC-F：5’-gaagaagactatgctcgtagatcagaag-3’。
SEQ ID NO.6：pspC-R：5’-ggagtagatggttgtgctggttt-3’。
the sequence of the TaqMan probe is as follows:
SEQ ID NO.7：
plo-probe：5’-CY5-cgagcctccatctccccgacg–BHQ1-3’
SEQ ID NO.8：
ICEMh-probe：5’-VIC-agccaaaccgccaacagagcca-BHQ1-3’
SEQ ID NO.9：
pspC-probe：
5’-FAM-aatataatcgcttgactcaacagcaaccgc-BHQ1-3’
it should be noted that the above primers and probes were synthesized by a method conventional in the art, and the primers and probes used in this example were both entrusted to the synthesis by Weichai fundamentals.
The instrument used for the real-time fluorescent PCR detection in the embodiment of the invention is a fluorescent quantitative PCR instrument ABI 7500.
1.2 based on the above primer, probe and system conditions, provides a triple real-time fluorescence quantitative PCR detection method for cryptobacter pyogenes, mannheimia haemolytica and streptococcus pneumoniae, comprising the following steps:
s1, extracting DNA as a reaction template;
the specific method for extracting the DNA comprises the following steps: after the bacterial strain is subjected to enrichment culture for 24h by using brain heart extract liquid, 1mL of bacterial liquid is taken for 12000r/min, the supernatant is centrifuged, and DNA is extracted by adopting a commercialized bacterial genome DNA extraction kit to be used as a template for reaction
S2, carrying out TaqMan triple real-time fluorescent quantitative PCR detection by adopting the primers and the probes.
The triple real-time fluorescence PCR detection reaction system is as follows: 25 μ L of: premix Ex TaqTM (2X) 12.5. mu.L, plo, ICEMh1, pspC primers, probe (10. mu.M) were 0.5. mu.L, ROX 0.3. mu.L, and deionized water 5.7. mu.L of DNA template 2. mu.L, respectively.
The reaction program of the triple real-time fluorescence PCR detection is Stage 1: pre-denaturation at 95 ℃ for 30 s; stage 2: 5s at 95 ℃, 34s at 60 ℃ and 40 cycles; and fluorescence signals were collected on three channels, FAM, VIC, Cy 5.
2. Specificity, sensitivity test, standard curve establishment and repeatability test
2.1 assay specificity
A large number of strains are adopted, the detection method established by the invention is respectively used for TaqMan triple real-time fluorescent PCR amplification, and the experimental strains are observed to have typical amplification curves. Some of the strains used in this experiment are listed in table 1.
TABLE 1 partial strain List for experiments
Experiments show that some bacteria, only, such as cryptococcus pyogenes, mannheimia haemolytica and streptococcus pneumoniae are exponentially amplified, and other bacteria are not generated by a typical amplification curve, so that the TaqMan triple real-time fluorescence PCR method for detecting virulence genes plo, ICEMh1 and pspC of the cryptococcus pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae, which is established by the invention, has good specificity, and is shown in figure 1. In FIG. 1, 1 is Streptococcus pneumoniae; 2 is cryptobacter pyogenes; 3 is mannheimia haemolytica; 4-17 are Salmonella typhimurium, Vibrio cholerae (Vb0) other than 01, Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus, Aeromonas hydrophila, Vibrio parahaemolyticus, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli, Listeria inokosa, Listeria illicii, and Pasteurella barnacii, respectively.
2.2 assay sensitivity
Cloning amplified sequences of cryptococcus pyogenes, mannheimia haemolytica and streptococcus pneumoniae in puc57(EcoRV, AmpR resistance) vector to construct a.p-m.h1-s.p recombinant plasmid DNA which is diluted to 1.25 × 10 by adding double distilled water6、1.25×105、1.25×104、1.25×103250, 50 and 10 copies/. mu.L, DNA was extracted for triple real-time fluorescent PCR detection.
The lowest detection quantitative limit of the invention can detect 20 copies of the standard substance positive plasmid, and the amplification result is shown in figure 2. In FIG. 2, 1 to 7 are respectively: 1.25X 106、1.25×105、1.25×104、1.25×103250, 50 and 10 copies/. mu.L of Cryptobacterium pyogenes, Mannheimia haemolytica and Streptococcus pneumoniae.
2.3 establishment of Standard Curve for multiplex real-time fluorescent quantitative PCR amplification of Cryptobacterium pyogenes, Mannheimia haemolytica and Streptococcus pneumoniae
The TaqMan triple real-time fluorescent PCR detection method established by the invention adopts 1.25 multiplied by 106、1.25×105、1.25×104、1.25×103250, 50 and 10 copies/. mu.L 7 concentrations of Cryptococcus pyogenes, Mannheimia haemolytica and Streptococcus pneumoniae plasmid DNA are subjected to triple real-time fluorescence quantitative PCR experiments, plo, ICEMh1 and pspC standard curves are respectively drawn, and the equations for obtaining the standard curves are respectively as follows:
m.h.:y＝3.5009x+40.844；
s.p:y＝3.3914x+40.418；
a.p:y＝3.4769x+40.746；
(m.h is Mannheimia haemolytica, s.p is Streptococcus pneumoniae, a.p is Cryptobacterium pyogenes)
The correlation coefficients of the standard curves are respectively 0.99, 0.99 and 0.99, and the amplification efficiencies are respectively 94%, 93% and 97%, which shows that the amplification efficiencies and linear correlation coefficients of the three genes of the detection system are good, and can meet the requirements of triple real-time fluorescent quantitative PCR detection, and the results are shown in FIG. 3. 2.4 triple real-time fluorescent quantitative PCR repeatability test
Cloning amplified sequences of cryptococcus pyogenes, mannheimia haemolytica and streptococcus pneumoniae in puc57(EcoRV, AmpR resistance) vector to construct a.p-m.h1-s.p recombinant plasmid, extracting template DNA and diluting into 1.25 × 106、1.25×105、1.25×104、1.25×103And 7 concentrations of 250, 50 and 10 copies/mu L, performing triple real-time fluorescence PCR detection, and performing 3 parallels on each dilution, wherein the result shows that the Ct value variation coefficients of three virulence genes are all below 2 percent, which indicates that the triple real-time fluorescence PCR detection method established in the method has good repeatability, stability and reliability. The results are shown in table 2:
TABLE 2TaqMan triple real-time fluorescent PCR repeatability test results
In table 2: m.h is Mannheimia haemolytica, s.p is Streptococcus pneumoniae, a.p is Cryptobacterium pyogenes.
2.5 quantitative determination of Cryptobacterium pyogenes, Mannheimia haemolytica and Streptococcus pneumoniae
Ct values of the corresponding channels obtained by real-time fluorescence PCR of the samples were obtained according to the standard curve equation of plo, ICEMh1, pspC: m.h. y 3.5009x +40.844, s.p: y 3.3914x +40.418 and a.p: y 3.4769x + 40.746; can calculate the log10(CFU) value of the concentration colonies of the cryptobacter pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae, thereby realizing the quantitative detection of the cryptobacter pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae.
In conclusion, the specific primer probes are designed according to the plo gene, the ICEMh1 gene and the pspC gene sequences of the cryptobacter pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae, the method for detecting the cryptobacter pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae by triple real-time fluorescent PCR is established, and the cryptobacter pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae can be quickly detected. The kit is suitable for rapid detection and epidemiological investigation of the distribution condition of virulence genes of the cryptococcus pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae of a large number of samples, and has positive significance for controlling the spread and treatment of diseases caused by the cryptococcus pyogenes, the mannheimia haemolytica and the streptococcus pneumoniae.
The invention optimizes the real-time fluorescent PCR detection system and reaction program conditions, successfully establishes a high-flux triple real-time fluorescent quantitative PCR detection method of the probes of cryptobacter pyogenes, mannheimia haemolytica and streptococcus pneumoniae TaqMan, has simple and safe operation, and adopts the recombinant plasmid a.p-m.h1-s.p as a standard sample to establish a standard curve at 1.25 x 106～1.0×101The copy/mu L range has good linear relation, and the lower limit of quantification can detect 20 copies of the standard positive plasmid. The whole amplification process is within 45min, has higher sensitivity, specificity and stability, can well meet the requirements of rapid detection and identification of cryptobacter pyogenes, mannheimia haemolytica and streptococcus pneumoniae in the breeding industry, can rapidly and accurately determine the contents of three bacteria in a sample, can analyze the carrying conditions of three virulence genes of the three bacteria, and can be used for epidemiological traceability and virulence intensificationAnd (4) weak analysis.
Sequence listing
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<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
agccaaaccg ccaacagagc ca 22
<210> 9
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
aatataatcg cttgactcaa cagcaaccgc 30
Claims (1)
1. A triple PCR detection kit for simultaneously detecting three pathogenic bacteria is characterized in that the three bacteria simultaneously detected are respectively Cryptobacterium pyogenes, Mannheimia haemolytica and Streptococcus pneumoniae;
the total volume of the reaction system in the kit is 25. mu.L, and the reaction system comprises:
2×Premix Ex TaqTM 12.5μL；
Plogene upstream primer 0.5 uL;
Plogene downstream primer 0.5 μ L;
Plogene probe 0.5 μ L;
ICEMh1gene upstream primer 0.5 uL;
ICEMh1gene downstream primer 0.5 μ L;
ICEMh1gene probe 0.5 μ L;
pspCgene upstream primer 0.5 uL;
pspCgene downstream primer 0.5 μ L;
pspCgene probe 0.5 μ L;
ROX 0.3μL ；
5.7 mu L of deionized water;
the DNA template was 2. mu.L;
wherein the content of the first and second substances,Ploa gene upstream primer,PloA gene downstream primer,PloA gene probe,ICEMh1A gene upstream primer,ICEMh1A gene downstream primer,ICEMh1A gene probe,pspCA gene upstream primer,pspCGene downstream primers andpspCthe concentration of the gene probes is 10 mu M;
wherein the content of the first and second substances,Ploa gene upstream primer,PloGene downstream primers andPlothe DNA sequences of the gene probes are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 7;
ICEMh1a gene upstream primer,ICEMh1Gene downstream primers andICEMh1the DNA sequences of the gene probes are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 8;
pspCa gene upstream primer,pspCGene downstream primers andpspCthe DNA sequences of the gene probes are respectively shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 9;
in the above-mentionedPloThe 5 'end of the gene probe is marked with a Cy5 fluorescent reporter group, and the 3' end of the gene probe is marked with BHQ 1; in the above-mentionedICEMh1The 5 'end of the gene probe is marked with a VIC fluorescent reporter group, and the 3' end of the gene probe is marked with BHQ 1; in the above-mentionedpspCThe 5 'end of the gene probe is marked with FAM fluorescent reporter group, and the 3' end is marked with BHQ 1.
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| CN113249503B (en) * | 2021-05-21 | 2022-06-28 | 华中农业大学 | LAMP primer group and method for detecting haemolytic mannheimia |
| CN114959083B (en) * | 2022-06-29 | 2023-08-22 | 西北农林科技大学 | Triple PCR detection primer set and kit |
| CN116004869A (en) * | 2022-12-30 | 2023-04-25 | 圣湘生物科技股份有限公司 | Composition for detecting staphylococcus haemolyticus, enterobacter cloacae and streptococcus pneumoniae |
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| BR112016012306A2 (en) * | 2013-12-03 | 2017-09-26 | Swiss Tropical And Public Health Inst | proline rich peptides for protection against s. pneumoniae |
| CN107964565A (en) * | 2017-12-08 | 2018-04-27 | 中国人民解放军总医院 | A kind of nucleic acid mass spectrometry method for being used to detect 10 kinds of clinical infection encountered pathogenic bacterias |
| CN108070665A (en) * | 2018-02-13 | 2018-05-25 | 林裕胜 | One kind is used to detect Mannheimia haemolytica real-time fluorescence PCR primer |
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