CN118186112A - PCR-HRM kit for rapidly identifying pullorum disease and salmonella gallinarum and application thereof - Google Patents
PCR-HRM kit for rapidly identifying pullorum disease and salmonella gallinarum and application thereof Download PDFInfo
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- 241000607132 Salmonella enterica subsp. enterica serovar Gallinarum Species 0.000 title claims abstract description 27
- 201000010099 disease Diseases 0.000 title description 20
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title description 20
- 241000607683 Salmonella enterica subsp. enterica serovar Pullorum Species 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 30
- 238000001514 detection method Methods 0.000 claims abstract description 22
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 3
- 241000293871 Salmonella enterica subsp. enterica serovar Typhi Species 0.000 claims description 16
- 108020004414 DNA Proteins 0.000 claims description 13
- 238000004458 analytical method Methods 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 8
- 239000007850 fluorescent dye Substances 0.000 claims description 7
- 229920006395 saturated elastomer Polymers 0.000 claims description 7
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 230000003321 amplification Effects 0.000 claims description 4
- 238000011880 melting curve analysis Methods 0.000 claims description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 238000012257 pre-denaturation Methods 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims 2
- 239000001046 green dye Substances 0.000 claims 2
- 238000007400 DNA extraction Methods 0.000 claims 1
- 238000002360 preparation method Methods 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 101150015947 fimH gene Proteins 0.000 abstract description 7
- 238000003745 diagnosis Methods 0.000 abstract description 3
- 230000000721 bacterilogical effect Effects 0.000 abstract description 2
- 238000002844 melting Methods 0.000 description 22
- 230000008018 melting Effects 0.000 description 22
- 241000607142 Salmonella Species 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 241000287828 Gallus gallus Species 0.000 description 6
- 235000013330 chicken meat Nutrition 0.000 description 6
- 238000011156 evaluation Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 108700028369 Alleles Proteins 0.000 description 3
- 208000035143 Bacterial infection Diseases 0.000 description 3
- 206010012735 Diarrhoea Diseases 0.000 description 3
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 3
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 3
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- FBPFZTCFMRRESA-GUCUJZIJSA-N galactitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-GUCUJZIJSA-N 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229960003104 ornithine Drugs 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
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- 102000036639 antigens Human genes 0.000 description 2
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- 238000011160 research Methods 0.000 description 2
- 201000008297 typhoid fever Diseases 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000007844 allele-specific PCR Methods 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 230000001684 chronic effect Effects 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
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- 230000004069 differentiation Effects 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
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- 244000144977 poultry Species 0.000 description 1
- 235000013594 poultry meat Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 208000013223 septicemia Diseases 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The invention discloses a kit for identifying PCR-HRM of salmonella pullorum and salmonella gallinarum and application thereof, wherein the kit comprises PCR-HRM primers, and the nucleotide sequences of the primers are as follows: the upstream primer fimH665-77F:5'-GGTCGTGGAGTTTGATTTCG-3'; the downstream primer fimH665-77R:5'-CGCCTTGCGGACGATTAC-3'. The invention establishes a PCR-HRM detection kit for rapidly identifying the salmonella pullorum and the salmonella gallinarum based on the fimH gene specific SNP locus, and the kit has the advantages of simple operation, high-throughput detection and the like, can obviously shorten the time required by identifying the salmonella pullorum and the salmonella gallinarum, has strong specificity and high sensitivity, has larger advantages in detection time and detection cost compared with the traditional bacteriological identification method, and is expected to have wide application prospect in rapid identification diagnosis of the salmonella pullorum and the salmonella gallinarum.
Description
Technical Field
The invention belongs to the technical field of pathogenic microorganism molecular detection, and particularly relates to a PCR-HRM kit for rapidly identifying pullorum disease and salmonella typhi and application thereof.
Background
Pullorum disease and fowl typhoid are important virulent bacterial infections of fowl, and two early bacterial diseases are found in veterinary communities, and the pathogens of the two bacterial diseases are salmonella pullorum and salmonella typhi respectively. Salmonella pullorum mainly causes acute systemic diseases of chickens, the death rate can reach more than 90%, while Salmonella pullorum causes acute or chronic septicemia, and mainly damages adult poultry.
Pullorum disease and salmonella gallinarum cannot express flagella, so that the salmonella gallinarum has no motility, and the size of colonies generated on an agar medium is obviously smaller than that of other serotypes of salmonella. In addition, since the pullorum disease and the salmonella gallinarum do not produce H 2 S, black metal luster colonies are not generated on most salmonella chromogenic mediums (such as SS, DHL, XLT, XLD, BS and the like), and colorless transparent microcolonies are generated, so that the pullorum disease and the salmonella gallinarum disease can be easily distinguished from other serotypes of salmonella by means of motility, colony size, colony color and the like. However, the identification difficulty of the salmonella pullorum and the salmonella gallinarum is high, because the 2 pathogens have many similarities in the aspects of surface antigen, clinical symptoms, quarantine purification and the like, so that the identification is difficult. Studies show that since pullorum disease and salmonella gallinarum all have the same antigen factor (1,9,12: -), the salmonella gallinarum is difficult to distinguish by the traditional salmonella diagnosis method (serological typing). At present, the biochemical fermentation tests of dulcitol and ornithine are mainly used for distinguishing, but the application of the biochemical fermentation tests is limited due to the fact that the biochemical fermentation tests cannot meet the requirements of rapid and batch detection.
With the rapid development of molecular diagnosis technology, a few molecular techniques for distinguishing pullorum disease and salmonella typhi are reported successively at present. For example, research shows that the I-type pilus adhesin gene (fimH) widely exists in chicken white diarrhea and chicken typhoid salmonella genomes, and the establishment and application of a chicken white diarrhea salmonella allele specific PCR detection method can be identified based on the phenomenon that alleles exist at 37 and 544 loci of fimH genes, such as KisielaD etal.Differentiation ofSalmonella GallinarumbiovarGallinarumfromSalmonella GallinarumbiovarPullorumbyPCR-RFLPofthefimHgene; Yao Yuan and the like. However, the applicant researches find that the 544 locus of the Salmonella typhi fimH gene is not specific (both A and T genotypes) and thus the nonspecific result is very easy to occur, so that an accurate identification method still needs to be further researched.
The applicant finds that a specific allele site exists at 665 in addition to 37 sites through the clone sequencing analysis of the preserved chicken white diarrhea and chicken salmonella typhi fimH genes. The inventor of the application successfully establishes a PCR-HRM method for rapidly identifying pullorum and salmonella gallinarum by utilizing a high-resolution melting curve (HRM, high-resolution melting) analysis technology, and realizes rapid, accurate and simple identification of the 2 pathogenic microorganisms.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the primary purpose of the invention is to provide a PCR-HRM primer for rapidly identifying pullorum and salmonella gallinarum.
The invention further aims at providing a PCR-HRM method for rapidly identifying pullorum disease and salmonella typhi. The method has the characteristics of simplicity and rapidness in operation, strong specificity, high sensitivity, high throughput and low cost, and can be widely applied to veterinary clinical detection.
The aim of the invention is achieved by the following technical scheme:
The PCR-HRM kit for rapidly identifying the pullorum disease and the salmonella gallinarum is characterized by comprising PCR-HRM primers, wherein the nucleotide sequences of the PCR-HRM primers are as follows:
The upstream primer fimH665-77F:5'-GGTCGTGGAGTTTGATTTCG-3';
The downstream primer fimH665-77R:5'-CGCCTTGCGGACGATTAC-3'.
A PCR-HRM method for rapidly identifying pullorum disease and salmonella typhi comprises the following steps:
S1: extracting bacterial genome DNA by using a commercialized kit to obtain a detection template;
S2: using bacterial genome DNA as a template, and performing an amplification reaction by using the upstream primer fimH665-77F, the downstream primer fimH665-77R and the saturated fluorescent dye to obtain an amplification product;
s3: HRM analysis was performed on the amplified products to distinguish pullorum or salmonella typhi.
Further, the PCR amplification reaction system described in step S2 is: each 10. Mu.l of the reaction solution contained 2X FAST EVAGREEN MASTER Mix 5. Mu.l, primer fimH665-77F 0.5. Mu.l, primer fimH665-77R 0.5. Mu.l, DNA template 1. Mu.l, ddH 2 O3. Mu.l.
The EVAGREEN MASTER Mix contains saturated fluorescent dye;
the detection primer concentration was 10. Mu.M.
Further, the amplification reaction procedure described in step S2 is: pre-denaturation at 95 ℃ for 2min, denaturation at 94 ℃ for 5s, annealing at 56 ℃ for 5s and extension at 72 ℃ for 25s, for 40 cycles; wherein the fluorescence signal acquisition temperature is 54 ℃ to 95 ℃ and the acquisition frequency is 0.3 ℃/step.
Further, the specific analysis process of HRM analysis in step S2 is as follows: when the Tm value of the sample to be detected is 81.43 +/-0.16 ℃, judging the sample to be detected as salmonella pullorum, and when the Tm value of the sample to be detected is 80.83 +/-0.15 ℃, judging the sample to be detected as salmonella gallinarum.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention reports a new allele locus of fimH genes for the first time, establishes a PCR-HRM method for rapidly identifying pullorum disease and salmonella gallinarum, has convenient operation, only needs to add saturated fluorescent dye into PCR reaction during detection, and simplifies the clinical examination flow.
(2) Compared with the traditional electrophoretic opening operation after PCR, the method realizes the real tube closing operation from the PCR process to the HRM analysis, and avoids cross contamination.
(3) The invention has strong specificity, can accurately distinguish pullorum disease and salmonella typhi, and has great advantages in detection time and detection accuracy compared with the traditional bacteriological typing method or other molecular detection methods.
(4) The invention has high sensitivity, the lowest detection concentration lower limit of the salmonella pullorum is 0.033pg, and the lowest detection concentration lower limit of the salmonella pullorum is 0.027pg.
Drawings
FIG. 1 is a HRM peak melting graph of pullorum disease, salmonella typhi identification primer screening experiments.
Fig. 2 is a HRM standardized melting graph of pullorum disease, salmonella typhi.
FIG. 3 is a peak melting graph of the sensitivity experiment for Salmonella pullorum and Salmonella typhi.
FIG. 4 is a peak melting graph of a pullorum disease, salmonella typhi specificity experiment.
Detailed Description
The present invention 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 invention, 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 invention falls within the scope of the present invention.
Example 1 screening of Salmonella pullorum and Salmonella typhimurium identification primers and establishment of PCR-HRM identification method
PCR-HRM discrimination primers were designed using 37-site (A.fwdarw.G) -specific Single Nucleotide Polymorphism (SNP) reported in the genes of Salmonella gallinarum fimH and 665-site (C.fwdarw.T) newly found by the applicant as subjects of study, respectively (Table 1).
TABLE 1 screening of PCR-HRM identification primers for Salmonella pullorum and Salmonella typhi
Preparing a PCR template: after the reference strains salmonella pullorum ATCC10398 and salmonella gallinarum ATCC9184 are subjected to broth enrichment, the genome DNA of the salmonella standard strain is extracted by using a kit and is used as a template to be detected.
The PCR-HRM reaction system was 10. Mu.l: 2X FAST EVAGREEN MASTER Mix 5. Mu.l, 0.5. Mu.l each upstream and downstream of the detection primer set, 1. Mu.l of DNA template, and ddH 2 O3. Mu.l.
The PCR-HRM reaction procedure was: pre-denaturation at 95 ℃ for 2min, denaturation at 94 ℃ for 5s, annealing at 56 ℃ for 5s and extension at 72 ℃ for 25s, for 40 cycles; wherein the fluorescence signal acquisition temperature is 54 ℃ to 95 ℃ and the acquisition frequency is 0.3 ℃/step.
The following results can be obtained in connection with fig. 1:
(1) fimH665-77 primer group has a melting temperature of Salmonella pullorum of 81.51deg.C, a melting temperature of Salmonella gallinarum of 80.87 deg.C, and a temperature difference of 0.67 deg.C;
(2) fimH665-130 primer groups have a melting temperature of 83.76 ℃ and a melting temperature of 83.44 ℃ for Salmonella pullorum, and a temperature difference of 0.32 ℃;
(3) fimH37 to 90 primer sets have a melting temperature of 83.76 ℃ and a melting temperature of 84.09 ℃ for Salmonella pullorum and a temperature difference of 0.33 ℃;
(4) fimH37 to 95 primer groups have a melting temperature of 83.76 ℃ and a melting temperature of 84.09 ℃ for Salmonella pullorum, and a temperature difference of 0.33 ℃;
The melting temperatures of Salmonella pullorum and Salmonella gallinarum in fimH-77 primer sets differ by 0.67 ℃, which is significantly better than that of the other 3 primer sets, and the 2 pathogens can be distinguished according to the standardized melting curve (figure 2) and the difference of Tm values.
Example 2 sensitivity test experiment
Sensitivity evaluation of the PCR-HRM discrimination method of the present invention was performed by the method of example 1. After enrichment of Salmonella pullorum ATCC10398 and Salmonella pullorum ATCC9184 with broth, bacterial genomic DNA was extracted using a commercial kit, 10-fold gradient dilution was performed after the original concentration was measured, and the DNA concentration was measured. Genomic DNA of different dilution gradients was subjected to sensitivity evaluation experiments. Analysis was performed according to the PCR amplification reaction and the melting curve analysis method of example 1, and the melting curve peaking chart is shown in FIG. 3. As can be seen from FIG. 3, the detection method provided by the invention has the lowest detection limit of 0.033pg for Salmonella pullorum and 0.027pg for Salmonella typhimurium, and has higher detection sensitivity.
Example 3 specificity evaluation experiment
The specificity evaluation of the PCR-HRM discrimination method of the present invention was performed by the method of example 1, and 6 Salmonella pullorum reference strains (ATCC 19945, ATCC10398, CMCC50771, CVCC519, CVCC521, CVCC 526) and 6 Salmonella pullorum reference strains (ATCC 9184, CVCC79301, CVCC536, CVCC537, CVCC538, 9R) were selected, and after overnight culture in broth, bacterial genomic DNA was extracted using a commercial kit and analyzed according to the PCR amplification reaction and melting curve analysis method of example 1. The melting curve peaking diagrams of different reference strains are shown in FIG. 4, wherein the melting temperature of Salmonella pullorum is 81.43 +/-0.16 ℃, the melting temperature of Salmonella gallinarum is 80.83 +/-0.15 ℃, the melting temperatures of two serotype strains are about 0.6 ℃, and the results are consistent with those of example 1, and the Salmonella pullorum and the Salmonella gallinarum can be distinguished according to the melting curve peaking and Tm value differences. The results show that the method established by the invention has better specificity.
Example 4 identification of clinical isolates
547 Clinical isolates of pullorum disease and salmonella gallinarum, which were stored in the subject group, were cultured overnight in broth, and bacterial genomic DNA was extracted by boiling, analyzed by PCR amplification reaction and melting curve analysis method of example 1, positive and negative controls were set, and simultaneously, identification was performed by using 2 biochemical experiments of ornithine and dulcitol. The results of the PCR-HRM method are completely consistent with the biochemical identification results of ornithine and dulcitol, and the established method has the advantages of simplicity, convenience, rapidness, high flux and the like, and has good specificity.
The invention and its embodiments have been described above by way of illustration and not limitation, and the invention 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 invention.
Claims (10)
1. A PCR-HRM kit for identifying salmonella pullorum and salmonella gallinarum, which is characterized by comprising PCR-HRM primers with the nucleotide sequences as follows:
The upstream primer fimH665-77F:5'-GGTCGTGGAGTTTGATTTCG-3';
The downstream primer fimH665-77R:5'-CGCCTTGCGGACGATTAC-3'.
2. The kit of claim 1, further comprising bacterial genomic DNA extraction reagents and saturated fluorescent dyes.
3. The kit of claim 2, wherein the saturated fluorescent dye is Eva green dye.
4. A PCR-HRM method for identifying salmonella pullorum and salmonella gallinarum, comprising the steps of:
S1: extracting bacterial genome DNA;
S2: performing PCR amplification by using the upstream primer fimH-77F, the downstream primer fimH665-77R and the saturated fluorescent dye according to claim 1 by using bacterial genome DNA as a template to obtain an amplification product;
s3: HRM analysis was performed on the amplified products to identify whether they were salmonella pullorum or salmonella typhimurium.
5. The PCR-HRM identification method as claimed in claim 4, wherein:
The PCR amplification reaction system in the step S2 is as follows: each 10. Mu.l of the reaction solution contained 2X FAST EVAGREEN MASTER Mix 5. Mu.l, primer fimH665-77F 0.5. Mu.l, primer fimH665-77R 0.5. Mu.l, DNA template 1. Mu.l, ddH 2 O3. Mu.l.
6. The PCR-HRM identification method as claimed in claim 4 or 5, wherein:
The saturated fluorescent dye is Eva green dye.
7. The PCR-HRM identification method as claimed in claim 4 or 5, wherein:
the detection primer concentration was 10. Mu.M.
8. The PCR-HRM identification method as claimed in claim 4, wherein:
The PCR-HRM analysis conditions in step S3 are as follows: pre-denaturation at 95 ℃ for 2min, denaturation at 94 ℃ for 5s, annealing at 56 ℃ for 5s and extension at 72 ℃ for 25s, for 40 cycles; wherein the fluorescence signal acquisition temperature is 54 ℃ to 95 ℃ and the acquisition frequency is 0.3 ℃/step.
9. The PCR-HRM identification method as claimed in claim 2, wherein:
And (3) performing high-resolution melting curve analysis on the PCR product obtained in the step (S2): when the Tm value is 81.43 +/-0.16 ℃, the salmonella pullorum is judged, and when the Tm value is 80.83 +/-0.15 ℃, the salmonella pullorum is judged.
10. Use of a PCR-HRM kit for the identification of salmonella pullorum and salmonella typhi according to any one of claims 1-3 for the preparation of a detection reagent for the identification of salmonella pullorum and salmonella typhi.
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