A primer set and kit for detecting Salmonella virulence genes based on fluorescent PCR technology

Abstract

This invention provides a primer set and kit for detecting Salmonella virulence genes based on fluorescent PCR technology, belonging to the field of pathogen detection technology. The invention provides a primer set for detecting Salmonella virulence genes based on fluorescent PCR technology, including primer sets for amplifying the ssaR, spvC, pefA, sipA, and fimA virulence genes, and primer sets for amplifying the sifA, sopE2, sopB, prgH, and stn virulence genes, respectively. Compared with other primer sets, this primer set not only accurately obtains the melting curve peaks of the target virulence genes, but also exhibits specificity in the melting curve peaks, with no overlap between pairs of melting curve peaks. It can accurately, specifically, and simultaneously detect the presence or absence of 10 Salmonella virulence genes. This invention provides a new method for rapidly detecting the virulence genotype of Salmonella.

Description

Technical Field

[0001] This invention belongs to the field of pathogen detection technology, specifically relating to a primer set and kit for detecting Salmonella virulence genes based on fluorescent PCR technology. Background Technology

[0002] Salmonella is a common Gram-negative bacterium belonging to the Enterobacteriaceae family that can cause zoonotic diseases, ranging from mild diarrhea to severe sepsis. Based on the clinical syndromes caused by Salmonella, it can be simply defined as two categories: typhoid Salmonella and non-typhoid Salmonella. Globally, the number of infections is high each year, and it can lead to death. In my country, the infection rate of Salmonella is also very high, causing cases of acute gastroenteritis, making Salmonella infection an increasingly serious public health problem. The pathogenicity of Salmonella is closely related to its virulence genes, and these virulence genes can be transferred between different Salmonella strains, potentially leading to enhanced pathogenicity in strains that acquire these genes. The typhoid epidemic that swept through my country in the 1980s was mainly caused by typhoid Salmonella acquiring the SPV virulence gene present in non-typhoid Salmonella. Therefore, studying the distribution and changes of Salmonella virulence genes is of great significance for the prevention, control, and treatment of Salmonella.

[0003] Currently, the detection of Salmonella virulence genes mainly relies on traditional PCR methods. Traditional PCR methods require electrophoresis to analyze PCR products, which is not only time-consuming and labor-intensive, but also highly susceptible to cross-contamination during the opening process. Fluorescent PCR technology, on the other hand, monitors changes in fluorescence signals in real time during PCR amplification, allowing for real-time detection of the PCR reaction. The entire process is completed in a closed tube, thus offering advantages such as simplicity, speed, high automation, and reduced risk of cross-contamination. Although fluorescent PCR technology is widely used in the detection and identification of pathogenic microorganisms, reports on its application to the detection of pathogenic microorganism virulence genes are scarce, and primers capable of simultaneously detecting ten major Salmonella virulence genes have not been reported. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a primer set for detecting Salmonella virulence genes based on fluorescent PCR technology, which can not only detect ten Salmonella virulence genes simultaneously, but also ensure that the primers for amplifying each virulence gene do not interfere with each other, thus guaranteeing the accuracy of the detection results.

[0005] The purpose of this invention is to provide a reagent kit for Salmonella virulence genes based on fluorescent PCR technology, which has the advantages of rapid detection and high degree of automation.

[0006] This invention provides a primer set for detecting Salmonella virulence genes based on fluorescent PCR technology, including primer set 1 and primer set 2;

[0007] Primer set 1 includes primers that amplify the virulence genes ssaR, spvC, pefA, sipA, and fimA, respectively.

[0008] Primer set 2 includes primers for amplifying the sifA, sopE2, sopB, prgH, and stn virulence genes, respectively.

[0009] The nucleotide sequences of each primer in primer set 1 are shown in SEQ ID NO:1 to SEQ ID NO:10; the nucleotide sequences of each primer in primer set 2 are shown in SEQ ID NO:11 to SEQ ID NO:20.

[0010] This invention provides a detection kit for Salmonella virulence genes based on fluorescent PCR technology, comprising the primer set and reagents for fluorescent PCR detection.

[0011] Preferably, the reagents for fluorescent PCR detection include EvaGreen fluorescent dye and / or KAPA 2G FastMultiplex PCR mixture.

[0012] This invention provides the application of the detection primer set in the preparation of a kit for detecting Salmonella virulence genotypes.

[0013] Preferably, the method for detecting the virulence genotype of Salmonella includes the following steps:

[0014] Using the DNA of the Salmonella sample as a template, reaction system 1 was prepared using primer set 1 from the primer set, and reaction system 2 was prepared using primer set 2 from the primer set.

[0015] The reaction system 1 and reaction system 2 were subjected to fluorescent PCR amplification. After the amplification was completed, the melting curve was monitored. The presence of the corresponding virulence gene in the sample was determined based on whether the primers for amplifying the virulence gene amplified the target melting curve peak.

[0016] This invention provides a primer set for detecting Salmonella virulence genes based on fluorescent PCR technology, comprising primer set 1 and primer set 2. Primer set 1 includes primers for amplifying the ssaR, spvC, pefA, sipA, and fimA virulence genes, respectively. Primer set 2 includes primers for amplifying the sifA, sopE2, sopB, prgH, and stn virulence genes, respectively. The nucleotide sequences of the primers in primer set 1 are shown in SEQ ID NO:1 to SEQ ID NO:10. The nucleotide sequences of the primers in primer set 2 are shown in SEQ ID NO:11 to SEQ ID NO:20. Compared with other primer sets designed for the same 10 target virulence genes, the detection primer set provided by this invention can not only accurately obtain the melting curve peaks of the target virulence genes, but also the melting curve peaks are specific, and there is no overlap between pairs of melting curve peaks. This enables accurate, specific, and simultaneous detection of the presence or absence of 10 Salmonella virulence genes, thus achieving rapid detection. Attached Figure Description

[0017] Figure 1 This is a melting curve peak diagram of the ssaR, spvC, pefA, sipA and fimA virulence genes detected by singleton PCR in Example 4 of the present invention;

[0018] Figure 2 This is a melting curve peak diagram of the sifA, sopE2, sopB, prgH and stn virulence genes detected by singleton PCR in Example 4 of the present invention;

[0019] Figure 3 This is a melting curve peak diagram of each virulence gene in reaction system 1 in Example 4 of the present invention;

[0020] Figure 4 This is a melting curve peak diagram of each virulence gene in reaction system 2 in Example 4 of the present invention;

[0021] Figure 5 This is a melting curve peak diagram of the ssaR virulence gene in Comparative Example 1 of this invention;

[0022] Figure 6 This is a melting curve peak diagram of the pefA virulence gene in Comparative Example 1 of this invention;

[0023] Figure 7 This is a melting curve peak diagram of the sifA virulence gene in Comparative Example 1 of this invention;

[0024] Figure 8 This is a melting curve peak diagram of the prgH virulence gene in Comparative Example 1 of this invention;

[0025] Figure 9This is a melting curve peak diagram of the ssaR virulence gene in Comparative Example 2 of this invention;

[0026] Figure 10 This is a melting curve peak diagram of the spvC virulence gene in Comparative Example 2 of this invention;

[0027] Figure 11 This is a melting curve peak diagram of the pefA virulence gene in Comparative Example 2 of this invention;

[0028] Figure 12 This is a melting curve peak diagram of the sipA virulence gene in Comparative Example 2 of the present invention;

[0029] Figure 13 This is a melting curve peak diagram of the fimA virulence gene in Comparative Example 2 of this invention;

[0030] Figure 14 This is a melting curve peak diagram of multiple virulence genes in Comparative Example 2 of the present invention;

[0031] Figure 15 This is a melting curve peak diagram of the sifA virulence gene in Comparative Example 2 of this invention;

[0032] Figure 16 This is a melting curve peak diagram of the sopE2 virulence gene in Comparative Example 2 of this invention;

[0033] Figure 17 This is a melting curve peak diagram of the sopB virulence gene in Comparative Example 2 of this invention;

[0034] Figure 18 This is a melting curve peak diagram of the prgH virulence gene in Comparative Example 2 of this invention;

[0035] Figure 19 This is a melting curve peak diagram of the stn virulence gene in Comparative Example 2 of the present invention;

[0036] Figure 20 This is a melting curve peak diagram of multiple virulence genes in Comparative Example 2 of the present invention. Detailed Implementation

[0037] This invention provides a primer set for detecting Salmonella virulence genes based on fluorescent PCR technology, including primer set 1 and primer set 2; primer set 1 includes primers for amplifying the virulence genes ssaR, spvC, pefA, sifA and sipA respectively; primer set 2 includes primers for amplifying the virulence genes fimA, sopE2, sopB, prgH and stn respectively, and the virulence genes corresponding to the amplified primers are shown in Table 1.

[0038] Table 1. Correspondence between amplification primers and virulence genes in this invention.

[0039]

[0040]

[0041] This invention provides a detection kit for Salmonella virulence genes based on fluorescent PCR technology, comprising the primer set and reagents for fluorescent PCR detection.

[0042] In this invention, the primer sets are each independently packaged with amplification primers for the 10 virulence genes. This invention does not impose any particular limitation on the source of the fluorescent PCR detection reagents; any fluorescent PCR detection reagents well-known in the art can be used. In embodiments of this invention, the fluorescent PCR detection reagents preferably include EvaGreen fluorescent dye and / or KAPA2GFast Multiplex PCR mixture.

[0043] This invention provides the application of the detection primer set in the preparation of a kit for detecting Salmonella virulence genotypes.

[0044] In this invention, the method for detecting the virulence genotype of Salmonella preferably includes the following steps:

[0045] Using the DNA of the Salmonella sample as a template, reaction system 1 was prepared using primer set 1 from the primer set, and reaction system 2 was prepared using primer set 2 from the primer set.

[0046] The reaction system 1 and reaction system 2 were subjected to fluorescent PCR amplification. After the amplification was completed, the melting curve was monitored. The presence of the corresponding virulence gene in the sample was determined based on whether the primers for amplifying the virulence gene amplified the target melting curve peak.

[0047] This invention does not impose any particular limitation on the method for extracting DNA from Salmonella samples; any bacterial DNA extraction method well-known in the art can be used. In this embodiment, the DNA from the Salmonella sample was extracted using the KAPA DNA Rapid Extraction Kit.

[0048] In this invention, the reaction system 1 is 20 μl: 1 μl DNA template, 1 μL EvaGreen fluorescent dye, 10 μl KAPA2G FastMultiplex PCR mixture, 0.4 μM ssaR-F, 0.4 μM ssaR-R, 0.4 μM spvC-F, 0.4 μM spvC-R, 0.3 μM pefA-F, 0.3 μM pefA-R, 0.1 μM sipA-F, 0.1 μM sipA-R, 0.1 μM fimA-F, 0.1 μM fimA-R, and the balance ddH2O;

[0049] Reaction system 2 consists of 20 μl: 1 μl DNA template, 1 μL EvaGreen fluorescent dye, 10 μl KAPA2G FastMultiplex PCR mixture, 0.4 μM sifA-F, 0.4 μM sifA-R, 0.2 μM sopE2-F, 0.2 μM sopE2-R, 0.2 μM sopB-F, 0.2 μM sopB-R, 0.05 μM prgH-F, 0.05 μM prgH-R, 0.1 μM stnF, 0.1 μM stn-R, with the remainder being ddH2O. The preferred reaction program for the fluorescent PCR amplification is: 95℃ for 3 min; 95℃ for 15 s, 60℃ for 30 s, 72℃ for 10 s, for 30 cycles. The preferred conditions for monitoring the melting curve are: holding at 78°C for 5 seconds, then increasing the temperature to 91°C at a rate of 0.1°C / s, with continuous detection of fluorescence intensity during the heating process. In this embodiment of the invention, the fluorescence PCR detection is performed on a Bio-Rad CFX96 fluorescence PCR instrument.

[0050] In this invention, the target melting curve peak is preferably as follows:

[0051] The virulence gene ssaR was amplified, and a melting curve peak with a Tm value of 80.7 ± 0.3℃ was obtained.

[0052] The virulence gene spvC was amplified, and a melting curve peak with a Tm value of 82.8 ± 0.2℃ was obtained.

[0053] The virulence gene pefA was amplified, resulting in a melting curve peak with a Tm value of 85.5 ± 0.1℃;

[0054] The virulence gene sipA was amplified, resulting in a melting curve peak with a Tm value of 87.4 ± 0.2℃;

[0055] The virulence gene fimA was amplified, resulting in a melting curve peak with a Tm value of 89.0 ± 0.2℃;

[0056] The virulence gene sifA was amplified, resulting in a melting curve peak with a Tm value of 79.6 ± 0.2℃;

[0057] The virulence gene sopE2 was amplified, and a melting curve peak with a Tm value of 81.8 ± 0.3℃ was obtained.

[0058] The virulence gene sopB was amplified, and a melting curve peak with a Tm value of 84.3 ± 0.2℃ was obtained.

[0059] The virulence gene prgH was amplified, resulting in a melting curve peak with a Tm value of 86.3 ± 0.1℃;

[0060] The virulence gene stn was amplified, and a melting curve peak with a Tm value of 88.1±0.3℃ was obtained.

[0061] In this invention, the Salmonella preferably includes at least one of the following Salmonella species: Salmonella enteritidis, Salmonella Londonii, and Salmonella typhimurium.

[0062] The following detailed description, in conjunction with embodiments, of a primer set and kit for detecting Salmonella virulence genes based on fluorescence PCR technology provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0063] Example 1

[0064] Design and artificial synthesis of a primer set for detecting Salmonella virulence genes based on fluorescent PCR technology

[0065] In this embodiment, 10 virulence genes—ssaR, sipA, fimA, sifA, sopE2, sopB, prgH, stn, spvC, and pefA—were used as target genes, and primers for fluorescent PCR detection were designed using Perimer 5.0 software. The design results are shown in Table 1.

[0066] Based on the nucleotide sequences of each primer pair, Suzhou Genewiz Biotechnology Co., Ltd. was commissioned to synthesize the primers.

[0067] Example 2

[0068] A Salmonella virulence gene detection kit based on fluorescent PCR technology includes the following components:

[0069] 1) Includes the primers synthesized in Example 1, specifically divided into two groups:

[0070] Primer set 1 includes primers that amplify the virulence genes ssaR, spvC, pefA, sipA, and fimA, respectively.

[0071] Primer set 2 includes primers for amplifying the sifA, sopE2, sopB, prgH, and stn virulence genes, respectively.

[0072] 2) EvaGreen fluorescent dye, purchased from Biotium;

[0073] 3) KAPA 2G Fast Multiplex PCR mixture, purchased from KAPA Biosystems.

[0074] Example 3

[0075] The method for detecting Salmonella virulence genes based on the kit described in Example 2 includes the following steps:

[0076] 1) Extract DNA from the Salmonella sample to be tested; the Salmonella sample to be tested has been amplified and verified using traditional PCR methods;

[0077] 2) Prepare two reaction systems, each with a total volume of 20 μl. Reaction system 1 consists of: 1 μl DNA template, 1 μL EvaGreen fluorescent dye, 10 μl KAPA 2G Fast Multiplex PCR mixture, 0.4 μM ssaR-F, 0.4 μM ssaR-R, 0.4 μM spvC-F, 0.4 μM spvC-R, 0.3 μM pefA-F, 0.3 μM pefA-R, 0.1 μM sipA-F, 0.1 μM sipA-R, 0.1 μM fimA-F, 0.1 μM fimA-R, and the remainder ddH2O.

[0078] Reaction system 2 consisted of 20 μl: 1 μl DNA template, 1 μL EvaGreen fluorescent dye, 10 μl KAPA 2G FastMultiplex PCR mixture, 0.4 μM sifA-F, 0.4 μM sifA-R, 0.2 μM sopE2-F, 0.2 μM sopE2-R, 0.2 μM sopB-F, 0.2 μM sopB-R, 0.05 μM prgH-F, 0.05 μM prgH-R, 0.1 μM stn-F, 0.1 μM stn-R, with the remainder being ddH2O.

[0079] 3) The two reaction systems prepared above were performed on a Bio-Rad CFX96 fluorescence PCR instrument. The reaction conditions were as follows: 95℃ pre-denaturation for 3 min, 95℃ for 15 s, 60℃ for 30 s, and 72℃ for 10 s for 30 cycles. Finally, the melting curve was monitored. After the last cycle was completed, the temperature was raised to 78℃, held for 5 s, and then gradually increased to 91℃ at a heating rate of 0.1℃ / s. During this heating process, the fluorescence intensity was continuously detected.

[0080] The presence and characteristics of melting curve peaks determine whether virulence genes are present. Virulence gene ssaR amplifies with a melting curve peak at a Tm value of 80.7 ± 0.3℃; virulence gene spvC amplifies with a melting curve peak at a Tm value of 82.8 ± 0.2℃; virulence gene pefA amplifies with a melting curve peak at a Tm value of 85.5 ± 0.1℃; virulence gene sipA amplifies with a melting curve peak at a Tm value of 87.4 ± 0.2℃; and virulence gene fimA amplifies with a melting curve peak at a Tm value of 8... The melting curve peaks were 9.0±0.2℃. The melting curve peaks for the virulence gene sifA (79.6±0.2℃), sopE2 (81.8±0.3℃), sopB (84.3±0.2℃), prgH (86.3±0.1℃), and stn (88.1±0.3℃) were observed. The results were consistent with those of conventional PCR detection.

[0081] The invention was validated using a previously reported conventional PCR method. Two strains of Salmonella Typhimurium and three strains of Salmonella Enteritidis were selected, and the results of the conventional PCR method were compared with those of the present invention. The results of the two methods were consistent.

[0082] Example 4

[0083] Methods for detecting the virulence genes of Salmonella typhimurium

[0084] This invention uses nine strains of Salmonella Typhimurium as experimental subjects. These nine strains were collected and summarized by a lower-level disease control center and identified using the national standard GB4789.4-2016. The results showed that all were Salmonella Typhimurium.

[0085] DNA was extracted from nine Salmonella Typhimurium strains. Using the DNA extracts from these nine strains as templates, singleton PCR was performed using the primers designed above (reaction conditions were the same as in Example 3). Figure 1 and Figure 2 The results are from single-primer amplification of the above 10 primer pairs.

[0086] Meanwhile, the method of Example 3 was used to retest nine strains of Salmonella Typhimurium. One strain of Salmonella Typhimurium was positive for ssaR, spvC, pefA, sipA, fimA, sifA, sopE2, sopB, prgH, and stn. The results are shown in [see attached table]. Figure 3 and Figure 4 Eight strains of Salmonella Typhimurium were positive for ssaR, sipA, fimA, sifA, sopE2, sopB, prgH, and stn, but negative for spvC and pefA.

[0087] Example 5

[0088] Methods for detecting the virulence genes of Salmonella London

[0089] This invention uses six strains of Salmonella London as experimental subjects. These six strains were collected and compiled by a lower-level disease control center and identified using the national standard GB4789.4-2016. The results showed that all were Salmonella London.

[0090] DNA was extracted from six Salmonella Londonis strains. Using the DNA extracts from these six strains as templates, the tests were performed according to the methods described above. Five strains of Salmonella Londonis were positive for ssaR, sipA, fimA, sifA, sopE2, sopB, prgH, and stn, but negative for spvC and pefA. One strain of Salmonella Londonis was positive for ssaR, sipA, fimA, sifA, sopE2, sopB, prgH, and stn, but negative for spvC, pefA, and sifA.

[0091] Example 6

[0092] Methods for detecting the virulence genes of Salmonella enteritidis

[0093] This invention uses 17 strains of Salmonella enteritidis as experimental subjects. The 17 strains of Salmonella enteritidis were collected and summarized by a lower-level disease control center, and identified according to the national standard GB4789.4-2016. The results showed that all of them were Salmonella enteritidis.

[0094] DNA was extracted from 17 Salmonella enteritidis strains. Using the Salmonella enteritidis DNA extract as a template, multiplex PCR was performed according to the detection method described in Example 3. Eleven Salmonella enteritidis strains were positive for ssaR, sipA, fimA, sifA, sopE2, sopB, prgH, and stn, but negative for spvC and pefA. Five Salmonella enteritidis strains were positive for ssaR, sipA, fimA, sopE2, sopB, prgH, and stn, but negative for spvC, pefA, and sifA. One Salmonella enteritidis strain was positive for ssaR, spvC, sipA, fimA, sopE2, sopB, prgH, and stn, but negative for pefA and sifA.

[0095] Comparative Example 1

[0096] In this experiment, 10 virulence genes—ssaR, sipA, fimA, sifA, sopE2, sopB, prgH, stn, spvC, and pefA—were used as target genes. Primers for fluorescent PCR detection were designed using Perimer 5.0 software. The virulence genes corresponding to the amplification primers are shown in Table 2.

[0097] Table 2. Correspondence between amplification primers and virulence genes in this invention.

[0098]

[0099] Based on the nucleotide sequences of each primer pair, Suzhou Genewiz Biotechnology Co., Ltd. was commissioned to synthesize the primers.

[0100] Following the method described in Example 3, the Salmonella Typhimurium strain with all positive virulence genes from Example 4 was used as a sample for testing.

[0101] Test results are shown Figures 5-8 When using the above primer combinations for fluorescent PCR amplification, some primers failed to amplify specific melting curve peaks, such as pefA (…). Figure 6 In addition, some primers amplified non-specific melting curve peaks, such as ssaR, prgH, and sifA. Figure 5 , Figure 7 and Figure 8 The results of virulence gene amplification were as follows: Primers for amplifying the stn gene were unstable; sometimes a melting curve peak appeared, and sometimes no amplification occurred. When a melting curve peak appeared, it was very low and did not form a typical specific melting curve peak. Not all primers designed using the target gene as a template can obtain a melting curve peak for the target gene.

[0102] Comparative Example 2

[0103] In this experiment, 10 virulence genes—ssaR, sipA, fimA, sifA, sopE2, sopB, prgH, stn, spvC, and pefA—were used as target genes. Primers for fluorescent PCR detection were designed using Perimer 5.0 software. The virulence genes corresponding to the amplification primers are shown in Table 3.

[0104] Table 3. Correspondence between amplification primers and virulence genes in this invention.

[0105]

[0106] Based on the nucleotide sequences of each primer pair, Suzhou Genewiz Biotechnology Co., Ltd. was commissioned to synthesize the primers.

[0107] The Salmonella typhimurium strain with all positive virulence genes from Example 4 was used for testing, following the method described in Example 3.

[0108] See results Figures 9-20 The test results showed that the primers amplified specific melting curve peaks. However, because the Tm values ​​of the melting curve peaks were too close together, overlap occurred during multiplex analysis, making it impossible to clearly determine the detection results of each virulence gene. Therefore, the above primer combination cannot meet the requirement of simultaneously detecting 10 virulence genes.

[0109] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer set for detecting Salmonella virulence genes based on fluorescent PCR technology, characterized by, The primer set comprises primer set 1 and primer set 2. The nucleotide sequences of the primers in the primer set 1 are shown in SEQ ID NO: 1-10, and the nucleotide sequences of the primers in the primer set 2 are shown in SEQ ID NO: 11-20.

2. A Salmonella virulence gene detection kit based on fluorescent PCR technology, characterized by, The primer set comprises the primer set in claim 1 and reagents for fluorescent PCR detection.

3. The test kit according to claim 2, characterized in that, The reagents for fluorescent PCR detection comprise EvaGreen fluorescent dye and / or KAPA 2G Fast Multiplex PCR mixture.

4. Application of the detection primer set in claim 1 in the preparation of a kit for detecting Salmonella virulence genotypes.

5. Use according to claim 4, characterized in that, The method for detecting Salmonella virulence genotypes comprises the following steps: Using the DNA of a Salmonella sample as a template, a reaction system 1 is prepared using primer set 1 in the primer set in claim 1, and a reaction system 2 is prepared using primer set 2 in the primer set in claim 1; The reaction system 1 and the reaction system 2 are subjected to fluorescent PCR amplification, and after the amplification is completed, a melting curve is monitored, and whether the sample contains the corresponding virulence gene is determined according to whether the virulence gene amplification primer amplifies the target melting curve peak.

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