A PCR-HRM kit for rapid identification of Salmonella pullorum and Salmonella gallinarum and its application

Abstract

The invention discloses a PCR-HRM kit for identifying Salmonella pullorum and Salmonella gallinarum and its application. The kit comprises PCR-HRM primers, and the primer nucleotide sequences are as follows: upstream primer fimH665-77F: 5'-GGTCGTGGAGTTTGATTTCG-3'; downstream primer fimH665-77R: 5'-CGCCTTGCGGACGATTAC-3'. The invention establishes a PCR-HRM detection kit for rapid identification of Salmonella pullorum and Salmonella gallinarum based on the specific SNP site of the fimH gene. The kit of the invention has the advantages of simple operation and high-throughput detection, and can significantly shorten the time required for identification of Salmonella pullorum and Salmonella gallinarum. At the same time, it has strong specificity and high sensitivity. Compared with traditional bacteriological identification methods, it has great advantages in detection time and detection cost, and is expected to have broad application prospects in rapid differential diagnosis of Salmonella pullorum and Salmonella gallinarum.

Description

Technical Field

[0001] The invention belongs to the technical field of pathogenic microorganism molecular detection, and particularly relates to a PCR-HRM kit for rapidly identifying pullorum and Salmonella gallinarum and application thereof. Background Art

[0002] Pullorum disease and fowl typhoid are two important bacterial infectious diseases of poultry, and are also two bacterial diseases discovered earlier by the veterinary community. Their pathogens are Salmonella pullorum and Salmonella gallisepticum. Salmonella pullorum mainly causes acute systemic diseases in chicks, with a mortality rate of more than 90%, while Salmonella gallisepticum causes acute or chronic septicemia, which mainly harms adult poultry.

[0003] Salmonella pullorum and Salmonella gallinarum cannot express flagella and therefore have no motility. The size of the colonies they produce on agar medium is significantly smaller than that of other serotypes of Salmonella. 2 S, so it does not produce black colonies with metallic luster on most Salmonella chromogenic culture media (such as SS, DHL, XLT4, XLD, BS, etc.), but produces colorless and transparent small colonies. Therefore, it is easy to distinguish Salmonella pullorum and Salmonella gallinarum from other serotypes of Salmonella by motility, colony size, colony color, etc. However, it is difficult to distinguish Salmonella pullorum and Salmonella gallinarum because these two pathogens have many similarities in surface antigens, clinical symptoms, quarantine and purification, etc., which makes it difficult to distinguish. Studies have shown that since Salmonella pullorum and Salmonella gallinarum have the same antigenic factors (1,9,12:-:-), it is difficult to distinguish them through traditional Salmonella diagnostic methods (serological typing). At present, it is mainly distinguished by biochemical fermentation tests of dactyl alcohol and ornithine, but its application is limited because it cannot meet the requirements of rapid and batch detection.

[0004] With the rapid development of molecular diagnostic technology, some molecular techniques for distinguishing Salmonella pullorum and Salmonella gallinarum have been reported. For example, studies have shown that type I fimbrial adhesin gene (fimH) is widely present in the genomes of Salmonella pullorum and Salmonella gallinarum. Based on the phenomenon that these two pathogens have alleles at sites 37 and 544 of the fimH gene, identification can be performed, such as KisielaD et al. Differentiation of Salmonella Gallinarum biovar Gallinarum from Salmonella Gallinarum biovar Pullorum by PCR-RFLP of the fimH gene; Yao Yuan et al., Establishment and application of Salmonella pullorum allele-specific PCR detection method. However, the applicant found that the site 544 of the fimH gene of Salmonella gallinarum is not specific (there are two genotypes, A and T), so non-specific results are very likely to occur, so further research on accurate identification methods is still needed.

[0005] The applicant analyzed the fimH gene clones and sequencing of the preserved Salmonella pullorum and Salmonella typhi, and found that in addition to site 37, there is also a specific allele site at site 665. The inventor of the present application successfully established a PCR-HRM method for rapid identification of Salmonella pullorum and Salmonella typhi using high-resolution melting (HRM) analysis technology, achieving rapid, accurate and simple identification of these two pathogenic microorganisms. Summary of the invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a PCR-HRM primer for rapid identification of Salmonella pullorum and Salmonella gallinarum.

[0007] Another object of the present invention is to provide a PCR-HRM method for rapid identification of Salmonella pullorum and Salmonella gallinarum. The method has the characteristics of simple and rapid operation, strong specificity, high sensitivity, high throughput and low cost, and can be widely used in veterinary clinical detection.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A PCR-HRM kit for rapid identification of Salmonella pullorum and Salmonella gallinarum, characterized in that the kit comprises PCR-HRM primers, the nucleotide sequences of which are as follows:

[0010] Upstream primer fimH665-77F: 5′-GGTCGTGGAGTTTGATTTCG-3′;

[0011] Downstream primer fimH665-77R: 5′-CGCCTTGCGGACGATTAC-3′.

[0012] A PCR-HRM method for rapid identification of Salmonella pullorum and Salmonella gallinarum comprises the following steps:

[0013] S1: Use a commercial kit to extract bacterial genomic DNA to obtain a detection template;

[0014] S2: using bacterial genomic DNA as a template, performing an amplification reaction using the upstream primer fimH665-77F, the downstream primer fimH665-77R and a saturated fluorescent dye described above to obtain an amplified product;

[0015] S3: Perform HRM analysis on the amplified products to distinguish between Salmonella pullorum and Salmonella gallinarum.

[0016] Furthermore, the PCR amplification reaction system described in step S2 is: each 10 μl reaction solution includes 2×FastEvaGreen Master Mix 5 μl, primer fimH665-77F 0.5 μl, primer fimH665-77R 0.5 μl, DNA template 1 μl, ddH 2 O 3μl.

[0017] The EvaGreen Master Mix contains saturated fluorescent dye;

[0018] The detection primer concentration is 10 μM.

[0019] Furthermore, the amplification reaction procedure described in step S2 is: pre-denaturation at 95°C for 2 min, denaturation at 94°C for 5 s, annealing at 56°C for 5 s, and extension at 72°C for 25 s, for a total of 40 cycles; wherein the fluorescence signal collection temperature is from 54°C to 95°C, and the collection frequency is 0.3°C / step.

[0020] Furthermore, the specific analysis process of the HRM analysis described in step S2 is: when the Tm value of the sample to be tested is 81.43±0.16°C, it is determined to be Salmonella pullorum, and when the Tm value is 80.83±0.15°C, it is determined to be Salmonella gallinarum.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention reports for the first time a new allele locus of the fimH gene and establishes a PCR-HRM method for rapid identification of Salmonella pullorum and Salmonella gallinarum. The method is easy to operate and only requires the addition of a saturated fluorescent dye to the PCR reaction during detection, thus simplifying the clinical testing process.

[0023] (2) Compared with the traditional open operation of electrophoresis after PCR, the present invention realizes a true closed-tube operation from the PCR process to the HRM analysis, thus avoiding cross contamination.

[0024] (3) The present invention has strong specificity and can accurately distinguish between Salmonella pullorum and Salmonella gallinarum. Compared with traditional bacteriological typing methods or other molecular detection methods, it has great advantages in detection time and detection accuracy.

[0025] (4) The present invention has high sensitivity, with the minimum detection concentration limit of Salmonella pullorum being 0.033 pg and the minimum detection concentration limit of Salmonella gallinarum being 0.027 pg. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the HRM peak-type melting curve diagram of the primer screening experiment for identifying Salmonella pullorum and Salmonella gallinarum.

[0027] Figure 2 This is the HRM standardized melting curve of Salmonella pullorum and Salmonella gallinarum.

[0028] Figure 3 It is a peak-type melting curve diagram of the sensitivity test of Salmonella pullorum and Salmonella gallinarum.

[0029] Figure 4 It is the peak-type melting curve diagram of the specific experiment of Salmonella pullorum and Salmonella gallinarum. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the embodiments and drawings. The following description is only a preferred embodiment of the present invention and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention is within the protection scope of the present invention.

[0031] Example 1 Screening of primers for identifying Salmonella pullorum and Salmonella gallinarum and establishment of PCR-HRM identification method

[0032] The 37 reported (A→G) specific single nucleotide polymorphisms (SNPs) in the fimH gene of Salmonella pullorum and Salmonella gallinarum and the 665 newly discovered (C→T) SNPs were used as research objects, and PCR-HRM identification primers were designed (Table 1).

[0033] Table 1. Screening of primers for PCR-HRM identification of Salmonella pullorum and Salmonella gallinarum

[0034]

[0035] Preparation of PCR template: After enrichment of reference strains Salmonella pullorum ATCC10398 and Salmonella gallinarum ATCC9184 in broth, genomic DNA of the above-mentioned Salmonella standard strains was extracted using a kit as a template to be tested.

[0036] The PCR-HRM reaction system was 10 μl: 2×Fast EvaGreen Master Mix 5 μl, 0.5 μl of upstream and downstream detection primers, 1 μl of DNA template, ddH 2 O 3μl.

[0037] The PCR-HRM reaction program was as follows: pre-denaturation at 95°C for 2 min, denaturation at 94°C for 5 s, annealing at 56°C for 5 s, and extension at 72°C for 25 s, for a total of 40 cycles; the fluorescence signal acquisition temperature was from 54°C to 95°C, and the acquisition frequency was 0.3°C / step.

[0038] Combination Figure 1 The following results are obtained:

[0039] (1) The melting temperature of Salmonella pullorum with the fimH665-77 primer set was 81.51°C, and the melting temperature of Salmonella gallinarum was 80.87°C, with a temperature difference of 0.67°C.

[0040] (2) The melting temperature of Salmonella pullorum in the fimH665-130 primer group was 83.76°C, and the melting temperature of Salmonella gallinarum was 83.44°C, with a temperature difference of 0.32°C.

[0041] (3) The melting temperature of Salmonella pullorum in the fimH37-90 primer group was 83.76°C, and the melting temperature of Salmonella gallinarum was 84.09°C, with a temperature difference of 0.33°C.

[0042] (4) The melting temperature of Salmonella pullorum in the fimH37-95 primer group was 83.76°C, and the melting temperature of Salmonella gallinarum was 84.09°C, with a temperature difference of 0.33°C;

[0043] The melting temperature difference between Salmonella pullorum and Salmonella gallinarum in the fimH665-77 primer set was 0.67℃, which was significantly better than the other three primer sets. Figure 2 ) and Tm value differences to distinguish these two pathogens.

[0044] Example 2 Sensitivity test experiment

[0045] The sensitivity evaluation of the PCR-HRM identification method of the present invention was performed using the method of Example 1. After Salmonella pullorum ATCC10398 and Salmonella gallinarum ATCC9184 were enriched in broth, bacterial genomic DNA was extracted using a commercial kit, and its original concentration was measured and then diluted 10 times in a gradient, and its DNA concentration was measured. The genomic DNA of different dilution gradients was subjected to a sensitivity evaluation test. The analysis was performed according to the PCR amplification reaction and melting curve analysis method of Example 1, and the melting curve peak type diagram is shown in FIG. Figure 3 As shown. Figure 3 It can be seen that the detection method of the present invention has a minimum detection limit of 0.033 pg for Salmonella pullorum and a minimum detection limit of 0.027 pg for Salmonella gallinarum, and has a high detection sensitivity.

[0046] Example 3 Specificity evaluation experiment

[0047] The specificity of the PCR-HRM identification method of the present invention was evaluated by the method of Example 1. Six reference strains of Salmonella pullorum (ATCC19945, ATCC10398, CMCC50771, CVCC519, CVCC521, CVCC526) and six reference strains of Salmonella gallinarum (ATCC9184, CVCC79301, CVCC536, CVCC537, CVCC538, 9R) were selected. 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 peak type of melting curves of different reference strains is shown in FIG. Figure 4 As shown, the melting temperature of Salmonella pullorum is 81.43±0.16°C, the melting temperature of Salmonella Gallinarum is 80.83±0.15°C, and the melting temperature difference between the two serotype strains is about 0.6°C. The results are consistent with those in Example 1, and Salmonella pullorum and Salmonella Gallinarum can be distinguished based on the melting curve peak type and Tm value difference. The above results show that the method established by the present invention has good specificity.

[0048] Example 4 Identification of Clinical Isolates

[0049] After 547 clinical isolates of Salmonella pullorum and Salmonella typhi preserved by the research group were cultured in broth overnight, the bacterial genomic DNA was extracted by boiling method, and analyzed according to the PCR amplification reaction and melting curve analysis method of Example 1, and positive control and negative control were set, and two biochemical experiments of ornithine and dulcitol were used for identification. As a result, 535 strains of Salmonella pullorum and 12 strains of Salmonella typhi were identified by PCR-HRM method, and the results were completely consistent with the biochemical identification results of ornithine and dulcitol, indicating that the method established by the present invention not only has the advantages of simplicity, rapidity, high throughput, etc., but also has good specificity.

[0050] The present invention and its embodiments are described schematically above, and the description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. Therefore, if a person skilled in the art is inspired by it and designs a structural method and an embodiment similar to the technical solution without creativity without departing from the purpose of the invention, they shall all fall within the protection scope of the present invention.

Claims

1. A PCR-HRM kit for identifying Salmonella pullorum and Salmonella gallinarum, characterized in that: The kit includes PCR-HRM primers, and the primer nucleotide sequences are as follows: Upstream primer fimH665-77F: 5′-GGTCGTGGAGTTTGATTTCG-3′; Downstream primer fimH665-77R: 5′-CGCCTTGCGGACGATTAC-3′.

2. The kit according to claim 1, further comprising a bacterial genomic DNA extraction reagent and a saturated fluorescent dye.

3. The kit according to claim 2, wherein the saturated fluorescent dye is Eva green dye.

4. A PCR-HRM method for distinguishing Salmonella pullorum from Salmonella gallinarum for non-diagnostic purposes, characterized in that The following steps are involved: S1: Extract bacterial genomic DNA; S2: Using bacterial genomic DNA as a template, performing PCR amplification using the upstream primer fimH665-77F, the downstream primer fimH665-77R and a saturated fluorescent dye in the PCR-HRM kit of claim 1 to obtain an amplified product; S3: Perform HRM analysis on the amplified product to identify whether it is Salmonella pullorum or Salmonella gallinarum.

5. The PCR-HRM method according to claim 4, characterized in that: The PCR amplification reaction system described in step S2 is: each 10 μl reaction solution includes 5 μl of 2×Fast EvaGreen MasterMix, 0.5 μl of primer fimH665-77F, 0.5 μl of primer fimH665-77R, 1 μl of DNA template, and 3 μl of ddH2O.

6. The PCR-HRM method according to claim 4 or 5, characterized in that: The saturated fluorescent dye is Eva green dye.

7. The PCR-HRM method according to claim 4 or 5, characterized in that: The concentration of the detection primer was 10 μM.

8. The PCR-HRM method according to claim 4, characterized in that: The HRM analysis conditions in step S3 are: pre-denaturation at 95°C for 2 min, denaturation at 94°C for 5 s, annealing at 56°C for 5 s, and extension at 72°C for 25 s, for a total of 40 cycles; wherein the fluorescence signal collection temperature is from 54°C to 95°C, and the collection frequency is 0.3°C / step.

9. The PCR-HRM method according to claim 4, characterized in that: The PCR product obtained in step S2 was subjected to high-resolution melting curve analysis: when the Tm value was 81.43±0.16° C., it was determined to be Salmonella pullorum; and when the Tm value was 80.83±0.15° C., it was determined to be Salmonella gallinarum.

10. Use of PCR-HRM primers for distinguishing Salmonella pullorum and Salmonella gallinarum in preparing a detection reagent for distinguishing Salmonella pullorum and Salmonella gallinarum, wherein the nucleotide sequence of the PCR-HRM primers is as follows: Upstream primer fimH665-77F: 5′-GGTCGTGGAGTTTGATTTCG-3′; Downstream primer fimH665-77R: 5′-CGCCTTGCGGACGATTAC-3′.

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