A primer-probe combination, kit, and method for detecting Salmonella pullorum in chickens based on fluorescence RT-RAA.

Abstract

This invention relates to the field of rapid detection technology for pathogenic microorganisms, specifically to a primer-probe combination, kit, and method of use for detecting Salmonella pullorum based on fluorescent RT-RAA. The primers for detecting Salmonella pullorum based on fluorescent RT-RAA are shown in SEQ ID NO.1 and SEQ ID NO.4. The kit developed using this primer-probe combination allows for rapid and efficient detection of Salmonella pullorum in samples using RT-RAA fluorescence, exhibiting high sensitivity and good repeatability, and showing promising application prospects in related rapid detection methods.

Description

Technical Field

[0001] This invention relates to the field of rapid detection technology for pathogenic microorganisms, specifically to a primer and probe combination, kit, and method of use for detecting Salmonella pullorum based on fluorescent RT-RAA. Background Technology

[0002] Salmonella pullorum (SP) is highly adaptable to specific hosts, spreading both horizontally and vertically, making it difficult to eradicate in poultry farms. It causes acute septicemia in chicks, primarily affecting those under 3 weeks old, with extremely high morbidity and mortality rates. As chickens age, their resistance to pullorum increases rapidly, and adult chickens can also be infected. It often leads to reduced egg production in hens, reproductive tract malformations, and a significant decrease in hatchability and chick emergence rates. Therefore, pullorum disease is one of the most important diseases threatening the healthy development of the poultry industry.

[0003] Currently, the main methods for preventing and controlling pullorum disease in poultry farms are quarantine culling combined with drug prevention. The commonly used method for detecting the disease is the plate agglutination method, which has a high false-positive rate and requires blood sampling from the chickens, causing irreversible damage or even death to chicks. Furthermore, the inappropriate use of antibiotics has led to a large number of drug-resistant bacteria, making complete eradication of the disease even more difficult. Laboratory testing requires high-precision PCR instruments and specialized technicians, which are difficult to achieve in grassroots poultry farms.

[0004] Recombinase-mediated isothermal nucleic acid amplification (RAA) technology utilizes the combined action of recombinase, single-strand binding proteins, and DNA polymerase to rapidly amplify nucleic acids by unwinding DNA double strands and extending primers to form double strands under isothermal conditions. RAA technology can complete the reaction and interpret results within 15-30 minutes at 39℃, offering advantages such as simplicity, speed, low equipment requirements, high sensitivity, and strong specificity. Currently, my country urgently needs a simple and accurate early detection method for Salmonella pullorum, as there is a pressing need for complete eradication of pullorum disease in chickens. Therefore, a rapid, simple, and accurate method for early detection and diagnosis of Salmonella pullorum is urgently required. Currently, there are no reports on the use of RAA for detecting Salmonella pullorum. Summary of the Invention

[0005] One of the objectives of this invention is to provide a primer for detecting Salmonella pullorum based on fluorescent 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-described 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 the fluorescence RT-RAA method, the primer comprising the primers shown in SEQ ID NO.1 and SEQ ID NO.4.

[0010] This invention also claims a kit for detecting Salmonella pullorum based on the fluorescence RT-RAA method, the kit comprising the aforementioned primers and probes. The probes include probes with the sequence shown in SEQ ID NO. 7, or probes modified based on the sequence shown in SEQ ID NO. 7.

[0011] Furthermore, the probe has four modification sites, with 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 quencher group BHQ1 (IBHQ1DT), modifying the space between the 29th and 32nd nucleotide T with tetrahydrofuran (THF) dSpacer, and modifying the 46th nucleotide (3' terminal nucleotide) with C3-spacer.

[0012] Preferably, the kit further includes 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 includes a positive control sample and a negative control sample.

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

[0015] S1: Add buffer A, 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 detection tube, positive control tube and negative control tube respectively. Finally add buffer B. After adding, immediately invert the reaction tube to mix thoroughly, centrifuge and finally put it into the fluorescence detection device.

[0016] S2: The fluorescence detection program is set as follows: react at a constant temperature of 39℃ for 30 minutes, and collect the fluorescence value of the FAM channel every 30 seconds;

[0017] S3: The results shall be interpreted as follows: if there is no amplification curve in the sample test tube, it indicates that there is no Salmonella pullorum in the test sample; if there is a typical amplification curve in the sample test tube, it indicates that there is Salmonella pullorum in the test sample.

[0018] More preferably, the amounts of each component in the kit are as follows: 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, the molar ratio of the upstream primer to the downstream primer in the primer probe 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-stranded binding protein, recA recombinase protein, Escherichia coli DNA polymerase, dithiothreitol, pyruvate kinase, DNA helicase gp41 protein, and reverse transcriptase.

[0022] This application also claims protection for the application of the probe or the kit described herein in the in vitro detection of the presence of Salmonella pullorum in samples for non-diagnostic purposes.

[0023] Compared with the prior art, the beneficial effects of this application are as follows:

[0024] This application provides primers for the detection of Salmonella pullorum in chickens using the RT-RAA fluorescence method, as shown in SEQ ID NO.1 and SEQ ID NO.4. A kit based on these primers was also developed. Using this kit, Salmonella pullorum in samples can be detected rapidly and efficiently using the RT-RAA fluorescence method, with high sensitivity and good repeatability, showing promising application prospects in related rapid detection methods. Attached Figure Description

[0025] Figure 1 The image shows the results of primer screening using the fluorescence method RT-RAA in Example 1. F1R1, F1R2, F1R3, F2R1, F2R2, F2R3, F3R1, F3R2, and F3R3 are nine primer combinations.

[0026] Figure 2 The results of the fluorescence-based RT-RAA specificity experiment in Example 1 are shown, where: Bacterial samples are bacterial samples.

[0027] Figure 3 The image shows the PCR results in Example 1, where 1-7 represent Salmonella pullorum, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus, Clostridium perfringens, and Salmonella typhimurium, respectively, and 8 is the negative control.

[0028] Figure 4 The image shows the results of the RT-RAA sensitivity test in Example 1, where the bacterial solution of Salmonella pullorum CVCC1800 was used as the standard. Different colors correspond to different concentrations of the standard, and NTC is nucleic acid-free water. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[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 provides a detailed description of the establishment of a fluorescence-based RT-RAA detection kit for Salmonella pullorum in chickens.

[0033] 1. Primer and probe design and synthesis: Using the conserved region of the *IpaJ* gene of *Salmonella pullorum* published in GenBank as the target site, and following the principles of RT-RAA primer design, sequence alignment analysis was performed using MegAlign software. Fragments with high homology were selected, and three pairs of primers and one probe were designed using Primer Premier 5, as shown in Table 1.

[0034] Table 1 Primers and probes

[0035]

[0036]

[0037] The SP-P mentioned in Table 1 above is a probe with four modification sites and a total length of 46 bases (as shown in SEQ ID No. 7). 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 space between the 29th and 32nd nucleotide T with tetrahydrofuran (THF) dSpacer, and modifying the 46th nucleotide (3' terminal nucleotide) with C3-spacer.

[0038] 2. Primer screening

[0039] Using Salmonella pullorum nucleic acid as a template, amplification was performed. After the reaction, the designed primers were screened based on the amplification curve, and the optimal primer pair was selected as F1R3. The selected optimal primer pair 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. Preparation of the reagent kit:

[0045] The nucleic acid detection kit of this embodiment includes: primer mixture, probe, Salmonella pullorum positive control sample, Salmonella pullorum negative control sample, 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 was performed using a fluorescent RT-RAA nucleic acid amplification kit. The total volume of the RT-RAA reaction system was 50 μL (Table 2). After preparation, the system was immediately transferred to a preheated 39°C real-time PCR instrument. The denaturation, annealing, and extension temperatures were all set to 39°C, with each cycle lasting 30 seconds, for a total of 60 cycles. Nuclease-free water was used as a negative control.

[0048] The three forward and three reverse primers of RT-RAA were combined into nine primer pairs, numbered 1-9 as F1 / R1, F1 / R2, F1 / R3, F2 / R1, F2 / R2, F2 / R3, F3 / R1, F3 / R2, and F3 / R3. Conventional RT-PCR was performed using these primer pairs. The amplified products were then sent to a sequencing database for sequencing to confirm if the sequences matched the ipaJ sequence. The nine primer pairs were then used to detect Salmonella pullorum at 39℃ for 30 minutes. Primers were screened based on fluorescence intensity 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] In Table 2, Abuffer is a hydrolysis buffer solution, 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 experiments of fluorescence-based RT-RAA

[0053] Use 4.9×10 8 -4.9×10 0 The sensitivity of this method was evaluated using serially diluted Salmonella pullorum suspensions at cfu / mL. The specificity of the method was tested using Salmonella pullorum, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus, Clostridium perfringens, and Salmonella typhimurium.

[0054] 6. Detection of clinical samples using fluorescence-based RT-RAA

[0055] Thirty fecal samples from one-day-old chicks and liver samples from dead embryos collected from a large poultry farm in Guangdong Province were pretreated and then tested by qPCR and RT-RAA fluorescence assay. The results showed that the positive detection rate of RAA was 13.3% (5 / 30), and the positive detection rate of qPCR was 16.6% (4 / 30). The concordance rate between RAA and qPCR results was 93.5%. Further analysis using SPSS software showed a Kappa value of 0.87 > 0.75 (P < 0.001), indicating that the established detection method has good consistency with national standards and a high concordance rate (Table 3).

[0056] Table 3. Concordance Rates of RAA and qPCR Clinical Sample Detection

[0057]

[0058] 7. Primer screening results

[0059] Among the nine primer pairs, SP-F1 and SP-R3 showed the highest fluorescence values ​​and the earliest peak in the reaction system. Figure 1 Subsequent experiments were conducted using a combination of SP-F1 and SP-R3 as primers for RT-RAA.

[0060] 8. Specificity and sensitivity test results

[0061] The RT-RAA fluorescence method only produced a fluorescent signal from Salmonella pullorum; other bacteria and nuclease-free water did not emit a fluorescent signal. Figure 2 RT-PCR only amplified the target band in *Salmonella pullorum*, while neither other bacteria nor nuclease-free water amplified any bands. Figure 3 ).from Figure 2 and Figure 3 As can be seen from the results, this method exhibits good specificity.

[0062] Using 4.9×10 8 -4.9×10 0 The sensitivity of the method was evaluated by serial dilutions of bacterial cultures at CFU / mL, with each dilution measured 8 times to assess the repeatability of the method (Table 4).

[0063] Table 4

[0064]

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

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

Claims

1. A kit for detecting Salmonella pullorum based on fluorescent method RT-RAA, characterized in that, The kit comprises primers and probes; the primers comprise primers as shown in SEQ ID NO. 1 and SEQ ID NO. 6, and the probes comprise probes of sequences as shown in SEQ ID NO.

7.

2. The kit for detecting Salmonella pullorum based on fluorescent RT-RAA according to claim 1, characterized in that, The probe has four modification sites in total, and the full length is 46 bases, and the modifications comprise: a FAM fluorescent group 6-carboxyfluorescein (I6FAMDT) is labeled on the 29th nucleotide T, a fluorescence quenching group BHQ1 (IBHQ1DT) is labeled on the 31st nucleotide T, the 29th nucleotide T and the 32nd nucleotide T are modified by tetrahydrofuran (THF) dSpacer, and the 46th nucleotide (3' terminal nucleotide) is modified by C3-spacer.

3. The kit of claim 1, wherein The kit further comprises RT-RAA dry powder reagent, A buffer and B buffer, the A buffer is a reaction buffer, and the B buffer is a magnesium acetate solution.

4. The kit of claim 3, wherein The kit further comprises a positive control sample and a negative control sample.

5. Use of the kit of claim 1 for detecting whether chicken white dysentery Salmonella exists in a sample in vitro, which is for non-diagnostic purposes.

6. Use according to claim 5, characterized in that, The final concentration of the primers in the reaction system is 10 μmol / L, and the final concentration of the probes in the reaction system is 10 μmol / L.

7. A method for detecting nucleic acid of chicken white dysentery Salmonella by using the kit of claim 4, comprising the following steps: S1: adding A buffer, primer mixture and probe into a reaction dry powder tube containing RT-RAA dry powder reagent, then adding a sample to be detected, chicken white dysentery Salmonella positive control sample and chicken white dysentery Salmonella negative control sample into a sample detection tube, a positive control tube and a negative control tube respectively, finally adding B buffer, and immediately after adding, inverting the reaction tube to fully mix, centrifuging, and finally placing into a fluorescence detection device; S2: the fluorescence detection program is set as: constant temperature 39℃ reaction for 30 min, and collecting fluorescence value of FAM channel every 30 s; S3: the result is interpreted according to the following judgment: if there is no amplification curve in the sample detection tube, it indicates that there is no chicken white dysentery Salmonella in the sample to be detected; if a typical amplification curve appears in the sample detection tube, it indicates that there is chicken white dysentery Salmonella in the sample to be detected.

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