Primer group for detecting salmonella pullorum based on RAA-LFD technology and application of primer group

Abstract

The invention belongs to the technical field of molecular biological detection, and particularly relates to a primer group for detecting salmonella pullorum based on an RAA-LFD technology and application of the primer group. The invention provides a primer group for detecting salmonella pullorum based on an RAA-LFD technology. The primer group comprises a forward primer, a reverse primer and a probe, the sequence of the forward primer is as shown in SEQ ID NO. 1; the sequence of the reverse primer is as shown in SEQ ID NO. 2; the sequence of the probe is based on SEQ ID NO.3, wherein the original base G of the 31st base of the SEQ ID NO.3 is replaced by an idSp group. According to the specific primer based on the RAA-LFD technology, the phenomenon of false positive caused by generation of primer dimers is avoided, the specificity is high, and the sensitivity is high. The primer group provided by the invention can be used for detecting salmonella pullorum and related strains, and only the salmonella pullorum is positive, so that the primer group shows good specificity.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biological detection, and particularly relates to a primer set for detecting Salmonella pullorum based on RAA-LFD technology and an application thereof. Background Art

[0002] Salmonella pullorum ( Salmonella pullorum Salmonella pullorum is the pathogen that causes pullorum. Discovered by Rettger in 1899, it lacks flagella and is non-motile. It is most severe in chicks under three weeks old, with morbidity and mortality rates reaching up to 90%. Infected chicks experience symptoms of white, sticky diarrhea, acute systemic infection, and septicemia. Infection in adult chickens primarily manifests as localized and chronic infections, resulting in symptoms such as abdominal drooping and decreased production performance. Vertical transmission is the primary route of Salmonella pullorum transmission, and systematic quarantine and decontamination of breeder chickens is key to controlling this pathogen.

[0003] Traditional bacterial isolation and culture methods are the gold standard for detecting Salmonella pullorum. However, they are time-consuming and labor-intensive, and cannot meet the needs of rapid detection of Salmonella pullorum in poultry products. Polymerase chain reaction (PCR) is used for the detection of Salmonella pullorum due to its high accuracy and relatively fast detection speed. However, this detection method requires an expensive thermal cycler, which limits its application in field conditions and grassroots farms. In addition, loop-mediated isothermal amplification (LMAP) has the characteristics of simple operation and high cost-effectiveness and has been developed for the detection of Salmonella pullorum. However, LMAP requires the design of 4 to 6 pairs of primers, which is relatively complex.

[0004] In summary, there is an urgent need to develop a new strategy for detecting Salmonella pullorum that is highly specific, sensitive, simple to operate, and fast and convenient to meet the urgent needs of the clinical frontline. Summary of the Invention

[0005] The purpose of the present invention is to provide a primer set for detecting Salmonella pullorum based on RAA-LFD technology, which solves the problems existing in the prior art.

[0006] To achieve the above purpose, the present invention provides the following solutions The present invention provides a primer set for detecting Salmonella pullorum based on RAA-LFD technology, wherein the primer set comprises a forward primer, a reverse primer and a probe; The sequence of the forward primer is shown in SEQ ID NO.1; The sequence of the reverse primer is shown in SEQ ID NO.2; The sequence of the probe is based on SEQ ID NO. 3, wherein the base at position 31 of SEQ ID NO. 3 is replaced by the idSp group, which is the original base G.

[0007] Preferably, the 5' end of the reverse primer contains a biotin label.

[0008] Preferably, the probe contains a 6-carboxyfluorescein label at its 5' end and a C3-Spacer phosphoramidite label at its 3' end.

[0009] A second aspect of the present invention provides a kit for detecting Salmonella pullorum, comprising the primer set and a lateral flow chromatography test strip.

[0010] The third aspect of the present invention provides the use of the primer set or the kit, wherein the use is for detecting and / or identifying Salmonella pullorum.

[0011] Preferably, the method for detecting and / or identifying Salmonella pullorum is: Obtain genomic DNA or bacterial solution of the sample to be tested; Using the genomic DNA or bacterial solution of the sample to be tested as a template, RAA-LFD amplification is performed using the kit; Determine whether it is Salmonella pullorum based on the test results of the lateral flow chromatography test strip.

[0012] Preferably, the conditions for RAA-LFD amplification are: Incubate the reaction at 38°C for 5 to 30 minutes.

[0013] Preferably, the incubation reaction time is 25 minutes.

[0014] Preferably, the final concentration of the forward primer in the RAA-LFD amplification system is 2.5 nM; The final concentration of the reverse primer in the RAA-LFD amplification system was 2.5 nM.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a primer set for detecting Salmonella pullorum based on the RAA-LFD technology, the primer set comprising a forward primer, a reverse primer, and a probe; the sequence of the forward primer is shown in SEQ ID NO.1; the sequence of the reverse primer is shown in SEQ ID NO.2; the sequence of the probe is based on SEQ ID NO.3, wherein the base at position 31 of SEQ ID NO.3 is replaced by the idSp group instead of the original base G. The specific primers based on the RAA-LFD technology provided by the present invention will not produce false positives due to the generation of primer dimers, and have strong specificity and high sensitivity. The primer set provided by the present invention can detect Salmonella pullorum and closely related strains, and only Salmonella pullorum is positive, showing good specificity.

[0016] The primer set of the present invention was used in experiments, and the detection limit of the genomic DNA of Salmonella pullorum reached 68.7 fg / μL, and the detection limit of the bacterial solution of Salmonella pullorum could reach 2.33×10 3 CFU / mL; the detection limit of conventional PCR for Salmonella pullorum genomic DNA is 68.7 pg / μL, and the detection limit for Salmonella pullorum is 2.33×10 4 CFU / mL, indicating that the detection sensitivity of the present invention for Salmonella pullorum is higher than that of ordinary PCR.

[0017] The primer set described in the present invention utilizes RAA-LFD technology to detect Salmonella pullorum, requiring minimal instrumentation. Amplification can be completed in a 20-minute incubation at a relatively low reaction temperature, such as 38°C. The amplified product can be detected using an LFD test strip in just 5 minutes, with results visible to the naked eye. The RAALFD rapid detection technology established by the present invention can serve as a simple method for real-time pathogen detection and rapid disease diagnosis, requiring no specialized personnel. It is suitable for rapid detection of Salmonella pullorum in resource-limited environments, such as aquaculture and the wild, and has promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the test result of Example 1, 1 is the test result of Example 1; NT is the negative control.

[0019] Figure 2 The following are the test results of Example 1 and Comparative Examples 1 to 5.

[0020] Figure 3 The test results of Example 1 to Example 6 are shown.

[0021] Figure 4 The results of specificity evaluation.

[0022] Figure 5 Figure 2 is the sensitivity evaluation result. A is the sensitivity result of RAA-LFD detection of genome; B is the sensitivity result of common PCR detection of genome; C is the sensitivity result of RAA-LFD detection of bacterial solution; D is the sensitivity result of common PCR detection of bacterial solution.

[0023] Figure 6 The results of the RAA-LFD method of the present invention on 19 clinical samples are shown. DETAILED DESCRIPTION

[0024] The present invention will be further described below by way of specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements fall within the scope of protection of the present invention.

[0025] The inventive concept of the present invention is as follows: Recombinase isothermal amplification (RAA), a novel isothermal nucleic acid amplification technology, is a simple, rapid, specific, sensitive, and economical molecular detection method for identifying a variety of pathogens. Currently, RAA-LFD, a method based on RAA and lateral flow chromatography (LFD) strips, has been successfully applied to the rapid detection of foodborne pathogens such as Vibrio vulnificus, Salmonella, and Staphylococcus aureus, enabling on-site diagnosis in remote areas such as the wild. However, RAA-LFD has not yet been developed for the detection of Salmonella pullorum. RAA-LFD is of great significance for the detection and epidemic control of Salmonella pullorum.

[0026] Compared to existing methods, RAA-LFD technology avoids the use of complex thermal cycling instruments and enables rapid, simple, and versatile nucleic acid test results. Its advantages, such as short reaction time, low reaction temperature, strong specificity, high sensitivity, and visual results, have made it highly popular in the field of on-site diagnosis.

[0027] In order to make those skilled in the art better understand the technical solution of the present invention and be able to implement it, the present invention is further described below in conjunction with specific examples. In the description of the present invention, if not otherwise specified, the reagents used are all commercially available and the methods used are all conventional techniques in the art.

[0028] The abbreviations of the present invention are shown in Table 1.

[0029] Table 1 Abbreviations Example 1 A primer set for detecting Salmonella pullorum based on RAA-LFD technology is as follows: The sequence of the Salmonella pullorum-specific gene seep17495 was downloaded from GenBank and used as a target for primer design. DNAMAN was used to search for conserved sequences. Based on the RAA primer design principles, Primer Premier 5 was used to design a pair of specific primers: forward primer Seep17495-F and reverse primer Seep17495-R. The 5' ends of the primers were biotin-labeled. Probes were then designed based on Seep17495-F and Seep17495-R. The probes were labeled with 6-carboxyfluorescein at the 5' end and C3-Spacer phosphoramidite at the 3' end, respectively. The G at base 31 of the probe was replaced with an idSp group.

[0030] At the same time, common pullorum primers SEEP400405-F and SEEP400405-R were designed as sensitivity controls for subsequent RAA-LFD.

[0031] seep17495 gene, GenBank accession number: CP012347.1:3688917-3689963.

[0032] The sequences of Seep17495-F and Seep17495-R are shown in SEQ ID NO.1 and SEQ ID NO.2; the probe sequence is shown in SEQ ID NO.3; the sequences of SEEP400405-F and SEEP400405-R are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0033] The above primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primer sequences are shown in Table 2.

[0034] Table 2 Primer sequences The RAA-LFD reaction was performed according to the instructions. The RAA nucleic acid amplification kit was purchased from Jiangsu Qitian Gene Biotechnology Co., Ltd. The present invention was slightly improved during use.

[0035] The present invention is carried out with a 50 μL reaction system, which is shown in Table 3, specifically as follows: Table 3 50 μL reaction system The reaction conditions are as follows: After lightly shaking and mixing the above substances, centrifuge. Place the centrifuge tube in a water bath at 38°C for 25 minutes to amplify. Take 10 μL of the amplified product and drop it onto the sample pad of the lateral flow chromatography test strip. Place the end of the sample pad of the lateral flow chromatography test strip vertically into a centrifuge tube containing 90 μL of buffer. After reacting for 5 minutes, read the result. Figure 1 As shown, red strips appear on both the C line and the T line, indicating that the test strip can successfully detect Salmonella pullorum.

[0036] In all figures of the present invention, the C line represents the control line; the T line represents the test line.

[0037] Because RAA primers differ from conventional PCR primers, being 1.5 to 2 times longer than conventional PCR primers, nonspecific amplification is highly likely to occur. Therefore, the present invention requires screening for appropriate primer concentrations to ensure that false positives are avoided and that the amplified bands are of appropriate brightness. Therefore, the present invention also provides Comparative Examples 1 to 5.

[0038] Comparative Example 1 to Comparative Example 5 A primer set for detecting Salmonella pullorum based on RAA-LFD technology is as follows: Comparative Examples 1 to 5 were all performed in 50 μL reaction systems. The concentrations of Seep17495-F and Seep17495-R primers in Comparative Examples 1 to 5 are shown in Table 4. The amounts of other substances and reaction conditions in the RAA-LFD reaction were exactly the same as in Example 1.

[0039] Table 4 Primer concentrations in different comparative examples From Example 1 and Comparative Examples 1 to 5, the results are shown in Figure 2 In a 50 μL reaction system, obvious false positives may occur at primer concentrations of 10 μmol / L and 5 μmol / L, while the reaction effect at a primer concentration of 1.25 μmol / L is the best and no false positives will occur, so 1.25 μmol / L is selected as the optimal reaction concentration for subsequent experiments.

[0040] Figure 2 1 to 6 are the experimental results of Comparative Example 1, Comparative Example 2, Example 1, Comparative Example 3 to Comparative Example 5, respectively; NT1 to NT6 are the negative controls of Comparative Example 1, Comparative Example 2, Example 1, Comparative Example 3 to Comparative Example 5, respectively.

[0041] Example 2 to Example 6 Examples 2 to 6 optimized the amplification time of RAA-LFD.

[0042] The reaction conditions of Example 2 to Example 6 are as follows: The reaction tube was incubated at 38° C. for 5 min, 10 min, 15 min, 20 min, and 30 min, respectively, and 10 μL of the amplified product was taken for test strip detection. The remaining conditions were exactly the same as in Example 1.

[0043] like Figure 3 The results show that the longer the reaction time, the more obvious the band on the test line of the test strip. The result can be observed after 10 minutes of reaction, the band is more obvious after 20 minutes of reaction, and the band effect is best after 25 minutes and 30 minutes of reaction. Therefore, 25 minutes is selected as the optimal reaction time.

[0044] Figure 3 1 to 6 are Example 2, Example 3, Example 4, Example 5, Example 1 and Example 6 respectively; NT is a negative control.

[0045] Example 7 An application of a primer set for detecting Salmonella pullorum based on RAA-LFD technology is as follows: 1. Evaluation of the specificity of the RAA-LFD method.

[0046] The RAA-LFD assay was performed under the optimal conditions of a 50 μL reaction volume, 2.1 μL of 1.25 μmol / L primers, and a 25-min reaction time to evaluate the specificity of the assay against two strains of Salmonella pullorum and seven other pathogenic bacteria. Information on the strains is provided in Table 5.

[0047] like Figure 4 As shown, two strains of Salmonella pullorum showed positive bands at the T line of the test strip, while the other seven pathogens did not show positive bands at the T line position of the test strip after amplification, indicating that the Salmonella pullorum RAA-LFD amplification primers designed based on the conserved sequence of the seep17495 gene are highly specific.

[0048] Figure 4 1 to 9 are Salmonella pullorum 2, Salmonella pullorum 1, Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, Salmonella and Listeria monocytogenes; NT is the negative control.

[0049] Table 5 Strain information Note: In Table 4, ATCC, American Type Culture Collection; “-” means no such item.

[0050] 2. RAA-LFD sensitivity evaluation.

[0051] 2.1. Genomic sensitivity evaluation.

[0052] The genomic DNA of Salmonella pullorum was serially diluted tenfold, and 2 μL of DNA was used for RAA-LFD detection under the optimal RAA reaction conditions to determine its detection limit and compare its sensitivity with that of conventional PCR method.

[0053] The sequences of the primers used in conventional PCR are shown in SEQ ID NO. 4 and SEQ ID NO. 5.

[0054] The sensitivity results of the RAA-LFD method of the present invention are shown in Figure 5 . Figure 5 A is the sensitivity result of RAA-LFD in detecting the genome of Salmonella pullorum; 1 to 7 represent the DNA concentrations of Salmonella pullorum of 6.87 ng / μL, 687 pg / μL, 68.7 pg / μL, 6.87 pg / μL, 687 fg / μL, 68.7 fg / μL, and 6.87 fg / μL, respectively; NT is the negative control; Figure 5 B is the sensitivity result of PCR detection of Salmonella pullorum genome. Lanes 1 to 7 represent Salmonella pullorum DNA concentrations of 6.87 ng / μL, 687 pg / μL, 68.7 pg / μL, 6.87 pg / μL, 687 fg / μL, 68.7 fg / μL and 6.87 fg / μL, respectively. NT is the negative control. M: DM600 DNA Marker.

[0055] like Figure 5 As shown in Figure A, the brightness of the T-line band gradually weakened as the concentration of S. pullorum genomic DNA decreased. Even when the genomic DNA concentration was as low as 68.7 fg / μL, positive T-line bands were still observed. Therefore, the detection limit of this method for S. pullorum is 68.7 fg / μL DNA.

[0056] The results of conventional PCR methods are as follows Figure 5 As shown in Figure B, as the concentration of Salmonella pullorum genomic DNA decreases, the brightness of the band gradually weakens. When the mass concentration of genomic DNA is as low as 6.87 pg / μL, a weak positive band can be observed.

[0057] The above results demonstrate that the sensitivity of the primer set of the present invention to the Salmonella pullorum genome is about 100 times that of conventional PCR.

[0058] 2.2. Sensitivity evaluation of pure bacterial solution The pure Salmonella pullorum solution was diluted ten-fold with physiological saline to obtain 2.33×10 5 CFU / mL~2.33×10 0CFU / mL of pure bacterial solution, 1 mL of pure bacterial solution with different concentrations was placed in a 1.5 mL centrifuge tube for RAA-LFD detection.

[0059] Figure 5 C is the sensitivity result of RAA-LFD detection of pure bacterial solution, 1 to 6 represent the concentration of pure bacterial solution of Salmonella pullorum of 2.33×10 5 CFU / mL, 2.33×10 4 CFU / mL, 2.33×10 3 CFU / mL, 2.33×10 2 CFU / mL, 2.33×10 1 CFU / mL and 2.33×10 0 CFU / mL, NT was the negative control. Figure 5 D is the sensitivity result of ordinary PCR detection of pure bacterial solution. Lanes 1 to 7 represent the concentration of pure Salmonella pullorum solution of 2.33×10 5 CFU / mL, 2.33×10 4 CFU / mL, 2.33×10 3 CFU / mL, 2.33×10 2 CFU / mL, 2.33×10 1 CFU / mL and 2.33×10 0 CFU / mL, NT is the negative control, M: DM600 DNA Marker.

[0060] like Figure 5 As shown in C, with the decrease of the concentration of pure Salmonella pullorum solution, the brightness of the T line gradually weakened. When the concentration of the solution was 2.33×10 2 CFU / mL, a faint positive band can be observed on the T line with the naked eye; when the bacterial concentration is 2.33×10 1 CFU / mL, no positive band appears on the T line; when the bacterial concentration is 2.33×10 3 CFU / mL, a clear positive band appeared on the T line. This indicates that the detection limit of this method for pure Salmonella pullorum liquid is 2.33×10 3 CFU / mL.

[0061] like Figure 5 As shown in D, with the decrease of the concentration of pure Salmonella pullorum solution, the brightness of the band gradually weakened. When the concentration of the solution was 2.33×10 4 CFU / mL, a faint positive band can be observed with the naked eye; when the bacterial solution concentration is 2.33×10 3 CFU / mL, no positive band appeared; this indicated that the detection limit of ordinary PCR for pure Salmonella pullorum solution was 2.33×104 CFU / mL.

[0062] The above results demonstrate that the sensitivity of the primer set of the present invention to the pure bacterial solution of Salmonella pullorum is 10 times that of conventional PCR.

[0063] In summary, the sensitivity of the amplification primer set for detecting Salmonella pullorum based on the RAA-LFD technology of the present invention is higher than the sensitivity of the amplification primer set for detecting Salmonella pullorum by conventional PCR.

[0064] 3. A method for detecting Salmonella pullorum based on RAA-LFD technology.

[0065] Nineteen 3-day-old chicks without specific immunity were selected and intraperitoneally injected with 10 8 Seven days after infection, livers were harvested and DNA extracted from the infected individuals. Feces were inoculated into brain heart infusion medium and cultured at 37°C, 180 rpm, for 12 hours, resulting in the isolation of 13 strains of Salmonella pullorum. The six strains listed in Table 5, including Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica, Salmonella typhimurium, and Staphylococcus epidermidis, were used as controls.

[0066] The bacterial suspension was centrifuged and genomic DNA was extracted. RAA-LFD detection was performed using the primer set and reaction conditions described in Example 1. The test results showed that 13 samples of Salmonella pullorum tested positive, while the other 6 samples tested negative. Figure 6 shown.

[0067] Figure 6 Among them, 1, 4, 7, 8, 11-19 are clinical samples isolated from chickens infected with Salmonella pullorum; 2, 3, 5, 6, 9 and 10 are Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella, Salmonella typhimurium and Staphylococcus epidermidis respectively; NT is the negative control.

[0068] The above examples illustrate that the amplification primer set for detecting Salmonella pullorum based on the RAA-LFD technology of the present invention has strong specificity and high sensitivity, and does not require DNA extraction. The method can detect Salmonella pullorum in a water bath reaction at 38°C for 25 minutes and a test strip test for 5 minutes. The method has a fast detection speed, low equipment requirements, and visual results, and has good application prospects in field testing.

[0069] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0070] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A primer set for detecting Salmonella pullorum based on RAA-LFD technology, characterized in that: The primer set includes a forward primer, a reverse primer and a probe; The sequence of the forward primer is shown in SEQ ID NO.1; The sequence of the reverse primer is shown in SEQ ID NO.2; The sequence of the probe is based on SEQ ID NO. 3, wherein the base at position 31 of SEQ ID NO. 3 is replaced by the idSp group, which is the original base G.

2. The primer set according to claim 1, wherein The 5' end of the reverse primer contains a biotin label.

3. The primer set according to claim 1, wherein The 5' end of the probe contains a 6-carboxyfluorescein label, and the 3' end contains a C3-Spacer phosphoramidite label.

4. A kit for detecting Salmonella pullorum, characterized in that: The kit comprises the primer set according to claim 1 and a lateral flow chromatography test strip.

5. Use of the primer set according to claim 1 or the kit according to claim 4, characterized in that: The application refers to detection and / or identification of Salmonella pullorum.

6. The use according to claim 5, characterized in that The method for detecting and / or identifying Salmonella pullorum is: Obtain genomic DNA or bacterial solution of the sample to be tested; Using the genomic DNA or bacterial solution of the sample to be tested as a template, RAA-LFD amplification is performed using the kit according to claim 4; Determine whether it is Salmonella pullorum based on the test results of the lateral flow chromatography test strip.

7. The use according to claim 6, characterized in that The conditions for the RAA-LFD amplification are: Incubate the reaction at 38°C for 5 to 30 minutes.

8. The use according to claim 7, characterized in that The incubation reaction time is 25 minutes.

9. The use according to claim 6, characterized in that The concentration of the forward primer in the RAA-LFD amplification system was 2.5 nM; The concentration of the reverse primer in the RAA-LFD amplification system was 2.5 nM.

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