Primer probe combination, kit and method for detecting salmonella pullorum based on fluorescence method RT-RAA

Abstract

The invention relates to the technical field of rapid detection of pathogenic microorganisms, in particular to a primer probe combination for detecting salmonella pullorum based on fluorescent RT-RAA, a kit and a use method. The primer for detecting salmonella pullorum based on the fluorescence method RT-RAA is as shown in SEQ ID NO. 1 and SEQ ID NO. 4. According to the kit developed by combining the primer with a probe, salmonella pullorum in a sample can be rapidly and efficiently detected by using an RT-RAA fluorescence method, and the kit is high in sensitivity and good in repeatability and has a good application prospect in development of related rapid detection.

Description

Technical Field

[0001] The present invention relates to the technical field of rapid detection of pathogenic microorganisms, and in particular to a primer-probe combination, a kit and a use method for detecting Salmonella pullorum based on fluorescent RT-RAA. Background Art

[0002] Salmonella pullorum (SP) is highly adaptable to a specific host. It can be transmitted horizontally and vertically, and is difficult to be removed and purified in poultry farms. It can cause acute sepsis in chicks, mostly affecting chicks under 3 weeks old, with extremely high morbidity and mortality rates. As the age of chickens increases, their resistance to pullorum increases rapidly, and adult chickens can also be infected, often leading to reduced egg production in hens, reproductive tract deformities, and a significant decrease in hatching and hatching rates. Therefore, pullorum is one of the important diseases that endanger the healthy development of the poultry industry.

[0003] At present, the main method for preventing and controlling pullorum in farms is quarantine elimination plus drug prevention. The method commonly used in farms to detect the disease is the plate agglutination method, which has a high false positive rate and requires blood sampling from chickens, causing irreversible damage or even death to chicks. In addition, due to the irrational use of antimicrobial drugs, a large number of drug-resistant bacteria are produced, making it more difficult to completely eliminate the disease. Laboratory testing requires high-precision PCR instruments and professional technicians, which is difficult to achieve in grassroots farms.

[0004] Recombinase-mediated isothermal nucleic acid amplification (RAA) technology uses the combined action of recombinase, single-strand binding protein and DNA polymerase to unwind the DNA double strands and extend the primers to form double strands under a constant temperature bar, thereby quickly achieving nucleic acid amplification. RAA technology can complete the reaction and interpret the results within 15-30 minutes at 39°C. It has the advantages of being simple, rapid, requiring low equipment, high sensitivity and strong specificity. At present, my country needs to thoroughly purify pullorum, and urgently needs an early detection method for Salmonella pullorum that is simple to operate and has high accuracy. Therefore, it is urgent to propose a method for detecting Salmonella pullorum that can be detected and diagnosed early, which is rapid, simple and accurate. There is currently no report on the use of RAA to detect Salmonella pullorum. Summary of the invention

[0005] One of the purposes of the present invention is to provide a primer for detecting Salmonella pullorum based on fluorescence RT-RAA.

[0006] Another object of the present invention is to provide a kit based on the above primers.

[0007] Another object of the present invention is to provide a method for detecting Salmonella pullorum based on fluorescent RT-RAA using the above kit.

[0008] To achieve the above technical objectives, the technical solution of the present invention is as follows:

[0009] A primer for detecting Salmonella pullorum based on fluorescence RT-RAA, the primer comprising primers as shown in SEQ ID NO.1 and SEQ ID NO.4.

[0010] The present invention also claims a kit for detecting Salmonella pullorum based on the fluorescence RT-RAA method, the kit comprising the above primers and probes. The probe comprises a probe with a sequence as shown in SEQ ID NO.7, or a probe modified on the basis of the sequence as shown in SEQ ID NO.7.

[0011] Furthermore, the probe has four modification sites and a total length of 46 bases, and the modifications include: labeling the 29th nucleotide T with the FAM fluorescent group 6-carboxyfluorescein (I6FAMDT), labeling the 31st nucleotide T with the fluorescent quencher group BHQ1 (IBHQ1DT), modifying the 29th nucleotide T and the 32nd nucleotide T with tetrahydrofuran (THF) dSpacer, and modifying the 46th nucleotide (3' terminal nucleotide) with C3-spacer.

[0012] Preferably, the kit further comprises RT-RAA dry powder reagent, A buffer and B buffer, wherein A buffer is a reaction buffer and B buffer is a magnesium acetate solution.

[0013] Preferably, the kit further comprises a positive control sample and a negative control sample.

[0014] The present application also provides a method for detecting Salmonella pullorum nucleic acid based on a kit, comprising the following steps:

[0015] S1: Add A buffer, primer mixture and probe to the reaction tube containing RT-RAA dry powder reagent, then add the sample to be tested, Salmonella pullorum positive control sample and Salmonella pullorum negative control sample to the sample test tube, positive control tube and negative control tube respectively, and finally add B buffer. After adding, turn the reaction tube upside down to mix thoroughly, centrifuge, and finally put it into the fluorescence detection equipment;

[0016] S2: The fluorescence detection program is set as follows: constant temperature at 39°C for 30 min, and the fluorescence value of the FAM channel is collected every 30 s;

[0017] S3: The result interpretation is carried out according to the following judgment: if there is no amplification curve in the sample test tube, it indicates that there is no Salmonella pullorum in the test sample; if a typical amplification curve appears in the sample test tube, it indicates that there is Salmonella pullorum in the test sample.

[0018] More preferably, the amounts of the components in the kit are: 2 μL of upstream primer, 2 μL of downstream primer, 0.3 μL of probe, 5 μL of Salmonella pullorum positive control sample, 5 μL of Salmonella pullorum negative control sample, 50 μL of RT-RAA dry powder reagent, 29.4 μL of A buffer and 2.5 μL of B buffer.

[0019] Preferably, in the primer probe, the molar ratio of the upstream primer to the downstream primer is 1:1.

[0020] Preferably, the concentration of the upstream primer is 10 μmol / L, the concentration of the downstream primer is 10 μmol / L, and the concentration of the probe is 10 μmol / L.

[0021] According to a preferred embodiment, the RT-RAA dry powder reagent comprises the following components: dNTPs, single-strand binding protein, recA recombinase protein, Escherichia coli DNA polymerase, dithiothreitol, pyruvate kinase, DNA helicase gp41 protein and reverse transcriptase.

[0022] The present application also claims to protect the use of the probe or the kit for in vitro detection of whether Salmonella pullorum exists in a sample, and the use is for non-diagnostic purposes.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] The present application provides a primer for detecting Salmonella pullorum based on the fluorescence method RT-RAA, as shown in SEQ ID NO. 1 and SEQ ID NO. 4. At the same time, a kit is obtained based on the primer, and the above kit can be used to quickly and efficiently detect Salmonella pullorum in a sample using the RT-RAA fluorescence method, and has high sensitivity and good repeatability, and has good application prospects in carrying out related rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a graph showing the results of the fluorescence RT-RAA primer screening in Example 1. F1R1, F1R2, F1R3, F2R1, F2R2, F2R3, F3R1, F3R2, and F3R3 are 9 primer combinations respectively.

[0026] Figure 2 The specific experimental results of RT-RAA by fluorescence method in Example 1, wherein: Bacterial samples are bacterial samples.

[0027] Figure 3 1-7 are the PCR result diagrams in Example 1, wherein 1-7 are Salmonella pullorum, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus, Clostridium perfringens and Salmonella typhimurium, and 8 is a negative control.

[0028] Figure 4 This is a graph showing the results of the fluorescence RT-RAA sensitivity experiment in Example 1, wherein the bacterial solution of the Salmonella pullorum standard bacterium CVCC1800 is used as the standard, wherein different color marks correspond to different concentrations of the standard, and NTC is nucleic acid-free water. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0031] It should be noted that the RT-RAA amplification kit in the following examples was purchased from Weifang Anpu Future Biotechnology Co., Ltd.; the primers in the following examples were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0032] Example 1 This example describes in detail the establishment of a fluorescent RT-RAA detection kit for Salmonella pullorum

[0033] 1. Design and synthesis of primers and probes: The conserved region of the ipaJ gene of Salmonella pullorum published in Genbank was used as the target site. According to the principle of RT-RAA primer design, MegAlign software was used for sequence alignment analysis, and fragments with high homology were selected. Three pairs of primers and a probe were designed using Primer Premier 5, as shown in Table 1:

[0034] Table 1 Primers and probes

[0035]

[0036]

[0037] The SP-P described in Table 1 above is a probe having four modification sites and a total length of 46 bases (as shown in SEQ ID No. 7), wherein the modifications include: labeling the 29th nucleotide T with a FAM fluorescent group 6-carboxyfluorescein (I6FAMDT), labeling the 31st nucleotide T with a fluorescent quenching group BHQ1 (IBHQ1DT), modifying the 29th nucleotide T and the 32nd nucleotide T with a tetrahydrofuran (THF) dSpacer, and modifying the 46th nucleotide (3' terminal nucleotide) with a C3-spacer.

[0038] 2. Primer screening

[0039] The Salmonella pullorum nucleic acid was used as a template for amplification. After the reaction was completed, the designed primers were screened according to the amplification curve, and the optimal primer pair was screened as F1R3. The optimal primer pair screened was renamed as follows:

[0040] Upstream primer (SEQ ID No.1):

[0041] SP-F:5'-TAATTCTTGCGGTGCATGTGCTTTGTTGGTTG-3'

[0042] Downstream primer (SEQ ID No.4):

[0043] SP-R:5'-TGCAACAACGATACCTTGAGGCATTGAATAACC-3'.

[0044] 3. Establishment of the kit:

[0045] The nucleic acid detection kit of this embodiment includes: a primer mixture, a probe, a positive control sample of Salmonella pullorum, a negative control sample of Salmonella pullorum, an RT-RAA dry powder reagent, Abuffer and B buffer, and nucleic acid-free water.

[0046] 4.RT-RAA system and primer screening

[0047] Fluorescent RT-RAA uses a fluorescent RT-RAA nucleic acid amplification kit. The total volume of the RT-RAA reaction system is 50 μL (Table 2). After the system is configured, it is immediately transferred to a fluorescent quantitative PCR instrument preheated to 39°C. The denaturation temperature, annealing temperature, and extension temperature are all set to 39°C, each cycle is 30 seconds, and 60 cycles are set. Nuclease-free water is used as a negative control.

[0048] The three forward primers and three reverse primers of RT-RAA were combined into 9 pairs of primers, 1-9 were F1 / R1, F1 / R2, F1 / R3, F2 / R1, F2 / R2, F2 / R3, F3 / R1, F3 / R2, F3 / R3. The combined primers were used for ordinary RT-PCR detection, and the amplified products were sent to Qingke for sequencing to compare whether the sequencing results were ipaJ sequences. The combined 9 pairs of primers were used to detect Salmonella pullorum, the reaction temperature was 39℃, the reaction time was 30min, and the primers were screened by fluorescence value and peak time.

[0049] Table 2 RT-RAA reaction system

[0050] Reagents concentration Volume / μL Abuffer - 29.4 Upstream primer 10μM 2 Downstream primer 10μM 2 Probe 10μM 0.6 <![CDATA[ddH2O]]> - 8.5 template - 5 Bbuffer - 2.5

[0051] Abuffer in Table 2 is a hydrolysis buffer, which is a 20% polyethylene glycol solution; B buffer is a magnesium acetate solution with a concentration of 280 mmol / L.

[0052] 5. Sensitivity and specificity experiment of fluorescence RT-RAA

[0053] With 4.9×10 8 -4.9×10 0 The sensitivity of the method was evaluated by serial dilution of Salmonella pullorum with cfu / mL. The specificity of the method was tested by using Salmonella pullorum, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus, Clostridium perfringens and Salmonella typhimurium.

[0054] 6. Fluorescence RT-RAA clinical sample detection

[0055] Thirty samples of one-day-old chick feces and dead embryo liver from a large poultry farm in Guangdong Province were pre-treated and then tested for qPCR and fluorescence RT-RAA. The results showed that the positive detection rate of RAA was 13.3% (5 / 30), the positive detection rate of qPCR was 16.6% (4 / 30), and the consistency rate of RAA and qPCR detection results was 93.5%. The Kappa value was further calculated and analyzed using SPSS software, and it was 0.87>0.75 (P<0.001), indicating that the established detection method was consistent with the national standard and had a high compliance rate (Table 3).

[0056] Table 3 RAA and qPCR clinical sample detection compliance table

[0057]

[0058] 7. Primer screening results

[0059] Among the 9 primer pairs, the fluorescence value in the reaction system of SP-F1 and SP-R3 was the largest, and the peak was early ( Figure 1 ). The combination of SP-F1 and SP-R3 was used as the primer for RT-RAA in subsequent experiments.

[0060] 8. Specificity and Sensitivity Experimental Results

[0061] In the fluorescence RT-RAA method, only Salmonella pullorum produced a fluorescent signal, while other bacteria and nuclease-free water did not emit a fluorescent signal ( Figure 2 ). Only Salmonella pullorum amplified the target band by RT-PCR, while other bacteria and nuclease-free water did not amplify the band ( Figure 3 ).from Figure 2 and Figure 3 As can be seen, the method system shows good specificity.

[0062] Use 4.9×10 8 -4.9×10 0 The sensitivity of the method was evaluated by serial dilution of bacterial solution with cfu / mL, and each dilution was measured 8 times to evaluate the repeatability of the method (Table 4).

[0063] Table 4

[0064]

[0065] The lowest detection limit of this method is 4.9 cfu / mL, indicating that the method has high sensitivity. It can be seen from Table 4 that the method has good repeatability.

[0066] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A primer for detecting Salmonella pullorum based on fluorescence RT-RAA, characterized in that: The primers include primers shown as SEQ ID NO.1 and SEQ ID NO.

4.

2. A kit for detecting Salmonella pullorum based on fluorescence RT-RAA, characterized in that: The kit comprises the primers and probes according to claim 1.

3. The kit for detecting Salmonella pullorum based on fluorescence RT-RAA according to claim 2, characterized in that: The probe includes a probe with a sequence as shown in SEQ ID NO.7, or a probe modified based on the sequence shown in SEQ ID NO.

7.

4. The kit for detecting Salmonella pullorum based on fluorescence RT-RAA according to claim 3, characterized in that: The probe has four modification sites and a total length of 46 bases. The modifications include: labeling the 29th nucleotide T with the FAM fluorescent group 6-carboxyfluorescein (I6FAMDT), labeling the 31st nucleotide T with the fluorescent quenching group BHQ1 (IBHQ1DT), modifying the 29th nucleotide T and the 32nd nucleotide T with tetrahydrofuran (THF) dSpacer, and modifying the 46th nucleotide (3' terminal nucleotide) with C3-spacer.

5. The kit according to claim 4, characterized in that The kit also includes RT-RAA dry powder reagent, Abuffer and B buffer, wherein the A buffer is a reaction buffer and the B buffer is a magnesium acetate solution.

6. The kit according to claim 4 or 5, characterized in that The kit also includes a positive control sample and a negative control sample.

7. Use of the primers according to claim 1 in detecting in vitro whether Salmonella pullorum exists in a sample, wherein the use is for non-diagnostic purposes.

8. Use of the kit according to claim 2 or 3 for detecting in vitro whether Salmonella pullorum exists in a sample, wherein the use is for non-diagnostic purposes.

9. The use according to claim 8, characterized in that: The final concentration of the primer in the reaction system is 10 μmol / L, and the final concentration of the probe in the reaction system is 10 μmol / L.

10. A method for detecting Salmonella pullorum nucleic acid using the kit according to claim 2, comprising the following steps: S1: Add A buffer, primer mixture and probe to the reaction tube containing RT-RAA dry powder reagent, then add the sample to be tested, Salmonella pullorum positive control sample and Salmonella pullorum negative control sample to the sample test tube, positive control tube and negative control tube respectively, and finally add B buffer. After adding, turn the reaction tube upside down to mix thoroughly, centrifuge, and finally put it into the fluorescence detection equipment; S2: The fluorescence detection program is set as follows: constant temperature at 39°C for 30 min, and the fluorescence value of the FAM channel is collected every 30 s; S3: The result interpretation is carried out according to the following judgment: if there is no amplification curve in the sample test tube, it indicates that there is no Salmonella pullorum in the test sample; if a typical amplification curve appears in the sample test tube, it indicates that there is Salmonella pullorum in the test sample.

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