CN116622872B - Salmonella gallinarum molecular detection kit and non-diagnostic detection method thereof - Google Patents
Salmonella gallinarum molecular detection kit and non-diagnostic detection method thereof Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
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- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/42—Salmonella
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Abstract
The application discloses a molecular detection kit for salmonella gallinarum and a non-diagnostic detection method thereof. The kit comprises a multiplex PCR detection primer group, a positive control and a negative control, wherein the positive control comprises salmonella typhimurium, salmonella pullorum and salmonella gallinarum genomic DNA; the negative control is sterilized double distilled water; the multiplex PCR detection primer set comprises: salmonella detection primer sequences SEQ ID NO.1, SEQ ID NO.2, chicken typhoid serotype detection primer sequences SEQ ID NO.3, SEQ ID NO.4, chicken typhoid biological detection primer sequences SEQ ID NO.5 and SEQ ID NO.6. The kit and the detection method have the advantages of rapidness, simpleness, strong specificity and high sensitivity, have larger advantages in detection time and detection cost compared with the traditional salmonella biochemical identification combined serological typing method, and are suitable for clinical batch detection.
Description
Technical Field
The application belongs to the technical field of molecular biological detection, and relates to a salmonella gallinarum molecular detection kit and a non-diagnostic detection method thereof.
Background
Salmonella is an important zoonotic primordium, mainly lodged in the intestinal tracts of humans and animals, and studies indicate that food poisoning caused by Salmonella is one of the highest proportion and the widest hazard among all bacterial food poisoning. The world has high-priority cases of food poisoning caused by salmonella infection all over the year, wherein the non-typhoid salmonella infection causes death of about 15.5 ten thousand people, 9380 ten thousand people are ill, and the annual incidence of Chinese is over 300 ten thousand. Investigation reports of global food-borne diseases by the world health organization in 2015 showed that salmonella incidence was first among 22 bacterial, viral and protozoal diseases.
Salmonella has the capability of vertical transmission and horizontal transmission, and creates serious challenges for the fields of poultry production, food safety and public health, and the salmonella is classified as an animal epidemic that is preferentially controlled and mainly prevented in the national medium-and-long-term animal epidemic control program (2012-2020). Studies have shown that poultry is the most important reservoir of Salmonella. Salmonella gallinarum can be classified into three categories, the first category is pullorum caused by pullorum biotype in salmonella gallinarum serotypes, the second category is fowl typhoid caused by fowl typhoid biotype in salmonella gallinarum serotypes, and the third category is fowl paratyphoid caused by other expressed flagella with motility (such as salmonella typhimurium, etc.). Because the three types of salmonellosis are different in epidemiology, pathological changes and prevention and control measures, accurate identification plays an important role in rapidly and effectively controlling epidemic diseases. Traditional identification methods employ bacteriology in combination with serological typing, but suffer from a number of drawbacks, such as: the method has the advantages of complicated and time-consuming operation steps (the steps of pre-enrichment, selective enrichment, biochemical identification and serological typing are needed in sequence), lower sensitivity and higher cost, and the chicken salmonellosis is seriously restricted by the reasons that chicken hakuri and chicken salmonella typhi have many similarities in etiology (are classified into 2 biotypes to which the same serological type belongs) and are further distinguished by specific biochemical indexes (such as ornithine, dulcitol and the like).
With the rapid development of molecular biology, the molecular detection method is continuously developed in practical inspection, and becomes one of the most potential detection methods for replacing the traditional detection methods. The multiple PCR technology has the advantages of sensitivity, specificity, simplicity, rapidness and the like of the PCR method, and simultaneously becomes one of the important technologies for simultaneously detecting multiple pathogens by the high efficiency. However, the design of multiplex PCR experiments is more complex than that of single PCR, so that the construction of a multiplex PCR reaction system is limited by considering the mutual interference among primers and the complicated optimization of main components and reaction conditions. In order to rapidly, accurately and simply identify different types of salmonella gallinarum, the application screens out specific detection targets by analysis according to known salmonella genome sequences, further designs multiplex PCR primers and develops a practical technology for detecting salmonella gallinarum.
Disclosure of Invention
Aiming at the defects of the traditional technology in identifying salmonella gallinarum, the application provides a molecular kit for specifically detecting salmonella gallinarum and a non-diagnostic detection method thereof.
The technical scheme of the application is as follows:
the application provides a salmonella gallinarum molecular detection kit and a non-diagnostic detection method thereof, wherein the salmonella gallinarum molecular detection kit comprises 10X PCR buffer solution, 2.5U/mul Taq DNA polymerase, 10Mm dNTPs, a multiplex PCR detection primer group, a positive control and a negative control, wherein the positive control comprises salmonella typhimurium ATCC14028, salmonella pullorum ATCC10398 and salmonella gallinarum ATCC9184 genome DNA; the negative control is sterilized double distilled water;
the multiplex PCR detection primer set comprises: salmonella detection primer sequences SEQ ID NO.1, SEQ ID NO.2, chicken typhoid serotype detection primer sequences SEQ ID NO.3, SEQ ID NO.4, chicken typhoid biological detection primer sequences SEQ ID NO.5 and SEQ ID NO.6.
Further, the multiplex PCR detection system comprises the following components: each 25. Mu.l of the reaction solution comprises 2.5. Mu.l of 10 XPCR buffer, 2. Mu.l of dNTPs, 0.25. Mu.l of Taq DNA polymerase, 3. Mu.l of detection primer group, 1-2. Mu.l of DNA template and a proper amount of sterilized double distilled water.
Further, the manufacturing method of the multiplex PCR detection method comprises the following steps: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s; annealing at 60 ℃ for 30s; extending at 72 ℃ for 25s; for a total of 30 cycles, and finally, the extension is carried out at 72 ℃ for 8min.
Further, the 10 XPCR buffer contains 100mM KCl, 80mM (NH) 4 )SO 4 100mM Tris-HCl, pH 9.0, 15mM MgCl 2 And 0.5% tergitol-type NP-40.
Further, the primer sequences are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.
Further, the primer concentrations were 10. Mu.M, and the detection primer sets were obtained after mixing in equal volumes.
Further, the dNTPs included dGTP, dCTP, dATP, dTTP, each component concentration was 2.5mM.
Further, a method for non-diagnostic detection using a salmonella gallinarum molecular detection kit, comprising the steps of:
s1: bacterial genomic DNA was extracted using a boiling method or commercial kit to obtain a detection template.
S2: multiplex PCR amplification, namely adding 10 times of PCR buffer solution, dNTPs, taq DNA polymerase, a detection primer group and a DNA template into a sterilization PCR reaction tube, adding sterilization double distilled water to the total volume of 25 mu l, setting positive and negative controls, and performing amplification reaction by using a PCR instrument; the amplified products were electrophoretically detected by 2% agarose gel electrophoresis and analyzed for the results.
Compared with the prior art, the application has the beneficial effects that:
the application can rapidly identify different types of salmonella gallinarum through one-time reaction, namely 1 stripe (383 bp) appears as salmonella causing fowl paratyphoid, 2 stripes (240bp+383 bp) appear as salmonella pullorum, and 3 stripes (240bp+383 bp+559 bp) appear as salmonella gallinarum. Compared with the traditional serological typing and common PCR detection, the application has great advantages in detection time and detection cost, and is suitable for batch detection. In particular, the primers of the present application can accurately distinguish pullorum disease from pullorum disease, and the related contents are also described in the "pullorum disease, pullorum disease and salmonella molecular identification kit and non-diagnostic detection method thereof" filed on the same day, the entire contents of which are incorporated into the present application and are part of the description of the present application, and the applicant has the right to modify the application file and reply to examination comments based on the contents described in the same day application.
Drawings
FIG. 1 is a gel electrophoresis display diagram of the multiplex PCR detection method in example 1. In the figure: m is DL-2000marker, lanes 1-3 are Salmonella paratyphi, salmonella pullorum and Salmonella gallinarum, respectively, and lane 4 is a negative control.
FIG. 2 is a graph showing the results of gel electrophoresis in the sensitivity evaluation experiment of the multiplex PCR detection method in example 3. In the figure: m is DL-2000marker, lanes 1-5 are samples of genomic DNA gradient dilutions of Salmonella typhimurium ATCC14028, and lane 6 is a negative control.
FIG. 3 is a graph showing the results of gel electrophoresis in the sensitivity evaluation experiment of the multiplex PCR detection method in example 3. In the figure: m is DL-2000marker, lanes 1-5 are Salmonella pullorum ATCC10398 genomic DNA gradient diluted samples, and lane 6 is a negative control.
FIG. 4 is a graph showing the results of gel electrophoresis in the sensitivity evaluation experiment of the multiplex PCR detection method in example 3. In the figure: m is DL-2000marker, lanes 1-5 are samples of Salmonella typhi ATCC9184 genomic DNA gradient dilutions, and lane 6 is a negative control.
Detailed Description
The present application is further described below with reference to the examples and drawings, which are given by way of illustration only, and not by way of limitation, of the preferred embodiments of the present application, and any person skilled in the art may make modifications to the equivalent embodiments using the technical matters disclosed above. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present application falls within the scope of the present application.
EXAMPLE 1 establishment of method for detecting Salmonella gallinarum molecules
Design of multiplex PCR primer: primers for specific detection of salmonella, salmonella gallinarum serotypes (including pullorum and salmonella gallinarum 2 biotypes) and salmonella gallinarum biotypes were screened after analysis and comparison according to the existing salmonella genomic DNA sequences in the GenBank database, as shown in table 1.
TABLE 1 Salmonella gallinarum molecular detection primers
The complete SEQ sequences of the salmonella detection primer sequences SEQ ID NO.1, SEQ ID NO.2, the chicken typhoid serotype detection primer sequences SEQ ID NO.3, SEQ ID NO.4, the chicken typhoid biological detection primer sequences SEQ ID NO.5 and SEQ ID NO.6 in the multiplex PCR detection primer group are shown in the sequence table.
Preparing a PCR template: salmonella typhimurium ATCC14028, salmonella pullorum ATCC10398 and Salmonella gallinarum ATCC9184 were each cultured in TSB broth medium for 12 hours, and the salmonella standard strain genome was extracted as a template to be examined using a commercial kit.
Preparation of multiplex PCR detection reagent: respectively diluting different primer pairs to 10 mu M concentration by using sterilized double steam, and mixing the primer pairs in equal volume to obtain a detection primer group;
multiplex PCR amplification reagents include: 10 XPCR buffer (its components include 100mM KCl, 80mM (NH 4) SO4, 100mM Tris-HCl pH 9.0, 15mM MgCl2 and 0.5% tergitol-type NP-40), dNTPs (containing dGTP, dCTP, dATP, dTTP, each component concentration of 2.5 mM), taq DNA polymerase (2.5U/. Mu.l), detection primer set.
Multiplex PCR detection system and amplification procedure: the detection system was 25. Mu.l, and specifically included: 10 XPCR buffer 2.5. Mu.l, dNTPs 2. Mu.l, taq DNA polymerase 0.25. Mu.l, detection primer set 3. Mu.l, DNA template 2. Mu.l and appropriate amount of sterile double distilled water. The amplification procedure includes the steps of: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 25s, 30 cycles total, and extension at 72 ℃ for 8min; after the reaction, the amplified product was taken out and stored at 4 ℃.
Determination of detection results of multiplex PCR method: mu.l of the amplified product was added to 1. Mu.l of 6 XLoadingbuffer, mixed well, spotted in a well of a 2% agarose gel electrophoresis plate, electrophoresed at 100V for 40min, and photographed under a gel imager for judgment. And (3) recovering the amplified product, cloning the amplified product into a pMD-19T vector, picking positive clone for sequencing, and comparing the result with a known sequence.
As shown in FIG. 1, M is DL-2000marker (2000 bp, 1000bp, 750bp, 500bp, 250bp and 100bp in this order), lanes 1 to 3 are Salmonella typhimurium ATCC14028 (383 bp), salmonella pullorum ATCC10398 (240bp+383 bp) and Salmonella gallinarum ATCC9184 (240bp+383 bp+559 bp), respectively, and lane 4 is a negative control.
EXAMPLE 2 specificity evaluation
The specificity evaluation of the multiplex PCR detection method of the present application was performed by the method of example 1, 656 experimental strains (46 strains of reference strain, 610 isolates) were selected, all experimental strains were subjected to enrichment culture, genomic DNA was extracted by boiling method, multiplex PCR detection was performed according to the method of example 2, and as shown in Table 2, all Salmonella gallinarum (3 reference strains and 12 isolates) amplified 3 specific amplified bands (240bp+383 bp+559bp), salmonella pullorum (3 reference strains and 524 isolates) amplified 1 specific amplified band (240bp+383 bp), other Salmonella serotypes (29 reference strains and 74 isolates) amplified 1 specific amplified band (383 bp), and all non-Salmonella reference strains (11 isolates) had no specific amplified band. The result shows that the method established by the application has better specificity. The strains and strain numbers used are shown in the following table, and "-" in the column of test results in the table indicates a negative result.
TABLE 2 specificity evaluation test results
Example 3 sensitivity test
Sensitivity evaluation of the multiplex PCR detection method of the present application was performed by the method of example 1. Salmonella typhimurium ATCC14028, salmonella pullorum ATCC10398 and Salmonella gallinarum ATCC9184 are respectively cultured in TSB broth culture medium for 12 hours, bacterial genome is extracted by using a commercial kit, the original concentration is measured, then the bacterial genome is mixed, 10-fold gradient dilution is carried out, and sensitivity evaluation tests are carried out on bacterial genome DNA of different dilution gradients. After the completion of the reaction, 5. Mu.l of the amplification product was mixed with 1. Mu.l of a 6 XLoading buffer, and the mixture was detected by agarose gel electrophoresis at a concentration of 2%. FIG. 2 shows the sensitivity test results of genomic DNA of Salmonella typhimurium ATCC14028, in which M is DL-200marker, lanes 1-6 are, in order, 126.3 ng/reaction, 12.63 ng/reaction, 1.263 ng/reaction, 126.3 pg/reaction, 12.63 pg/reaction, 1.263 pg/reaction), and lane 7 is a negative control; FIG. 3 shows the sensitivity test results of genomic DNA of Salmonella pullorum ATCC10398, in which M is DL-200marker, lanes 1-6 are in the order of 90.8 ng/reaction, 9.08 ng/reaction, 908 pg/reaction, 90.8 pg/reaction, 9.08 pg/reaction, 0.908 pg/reaction), and lane 7 is a negative control; FIG. 4 shows the results of the genomic DNA sensitivity test of Salmonella typhi ATCC9184, in which M is DL-200marker, lanes 1-5 are 142.7 ng/reaction, 14.27 ng/reaction, 1.427 ng/reaction, 142.7 pg/reaction, 14.27 pg/reaction, 1.427 pg/reaction, and lane 7 is a negative control. As shown in FIGS. 2-4, the molecular detection method provided by the application has higher detection sensitivity to the genomic DNA of salmonella typhimurium ATCC14028, salmonella pullorum ATCC10398 and salmonella gallinarum ATCC9184, namely 12.63 pg/reaction, 9.08 pg/reaction and 14.27 pg/reaction.
Example 4 Assembly of Salmonella gallinarum molecular detection kit
Extracting salmonella standard strain genome DNA (comprising salmonella typhimurium ATCC14028, salmonella pullorum ATCC10398 and salmonella gallinarum ATCC 9184) by using a kit to obtain a positive control of the application; 3 pairs of specific detection primers are synthesized according to the sequences in the table in the example 1, and the detection primers are diluted to 10 mu M concentration by sterilized double distilled water and mixed in equal volume to obtain a detection primer group; multiplex PCR amplification reagents: 10 XPCR buffer (its components include 100mM KCl, 80mM (NH 4) SO4, 100mM Tris-HCl pH 9.0, 15mM MgCl) 2 And 0.5% tergitol-type NP-40), dNTPs (containing dGTP, dCTP, dATP, dTTP at a concentration of 2.5mM each component), taq DNA polymerase (2.5U/. Mu.l), a detection primer set, a positive control (Salmonella standard strain DNA template), and a negative control (sterilized double distilled water).
The salmonella gallinarum molecular detection kit is assembled by packaging the above related reagents and products together and then matching with product use instructions (comprising product preservation conditions, reaction procedures, result judging methods and the like).
The application and its embodiments have been described above by way of illustration and not limitation, and the application is illustrated in the accompanying drawings and described in the drawings in which the actual structure is not limited thereto. Therefore, if one of ordinary skill in the art is informed by this disclosure, the structural mode and the embodiments similar to the technical scheme are not creatively designed without departing from the gist of the present application.
Claims (7)
1. A salmonella gallinarum molecule detection kit is characterized in that: comprises multiple PCR detection primer groups, positive control and negative control, wherein the positive control comprises salmonella typhimurium, salmonella pullorum and salmonella gallinarum genome DNA; the negative control is sterilized double distilled water;
the multiplex PCR detection primer set comprises: salmonella detection primer sequences SEQ ID NO.1, SEQ ID NO.2, chicken typhoid serotype detection primer sequences SEQ ID NO.3, SEQ ID NO.4, chicken typhoid biological detection primer sequences SEQ ID NO.5 and SEQ ID NO.6.
2. The salmonella gallinarum molecular detection kit of claim 1, wherein: the positive controls were salmonella typhimurium ATCC14028, salmonella pullorum ATCC10398, and salmonella gallinarum ATCC9184 genomic DNA.
3. The salmonella gallinarum molecular detection kit of claim 1, wherein: the multiplex PCR detection system comprises the following components: each 25. Mu.l of the reaction solution comprises 2.5. Mu.l of 10 XPCR buffer, 2. Mu.l of dNTPs, 0.25. Mu.l of Taq DNA polymerase, 3. Mu.l of detection primer group, 1-2. Mu.l of DNA template and a proper amount of sterilized double distilled water.
4. The salmonella gallinarum molecular detection kit of claim 1, wherein: the multiplex PCR detection primer group comprises primers shown in SED ID NO 1, 2, 3, 4, 5 and 6.
5. The salmonella gallinarum molecular detection kit of claim 1, wherein: the concentration of the detection primer is 10 mu M, and the detection primer group is obtained after equal volume mixing.
6. The salmonella gallinarum molecular detection kit of claim 1, wherein: the dNTPs included dGTP, dCTP, dATP, dTTP, each component at a concentration of 2.5mM.
7. A method for non-diagnostic detection using a salmonella gallinarum molecular detection kit, comprising the steps of:
s1: extracting bacterial genome DNA by using a boiling method or a commercial kit to obtain a detection template;
s2: multiplex PCR amplification, namely adding 10 times of PCR buffer solution, dNTPs, taq DNA polymerase, a detection primer group and a DNA template into a sterile PCR reaction tube, adding sterilized double distilled water to the total volume of 25 mu l, setting positive and negative controls, and performing amplification reaction by using a PCR instrument; electrophoresis detection is carried out on the amplified product by adopting 2% agarose gel electrophoresis, and the result is analyzed;
the detection primer set includes: salmonella detection primer sequences SEQ ID NO.1, SEQ ID NO.2, chicken typhoid serotype detection primer sequences SEQ ID NO.3, SEQ ID NO.4, chicken typhoid biological detection primer sequences SEQ ID NO.5 and SEQ ID NO.6.
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