A rapid method for identification of canine brucella

Abstract

The application discloses a rapid identification method for canine Brucella, and relates to the technical field of molecular detection. The rapid identification method comprises the following steps: extracting genomic DNA of a to-be-detected sample; using the genomic DNA as a template, performing fluorescence quantitative detection by using a primer and a probe combination, collecting signals of a fluorescence channel, and identifying whether the to-be-detected sample is canine Brucella. wbdA By screening a specific SNP site of canine Brucella, designing a specific primer and probe combination, and constructing a multi-channel fluorescence quantitative detection system, the primer and the probe combination comprise Brucella universal primer and probe and species-specific primer and probe, synchronous detection is realized by different fluorescence labels, Brucella infection can be rapidly confirmed, canine Brucella can be accurately distinguished from other Brucella, an efficient tool is provided for rapid identification of canine Brucella, and the method has important practical values for epidemiological prevention and control of brucellosis and prevention of zoonosis.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology, and in particular to a rapid identification method for Brucella canis. Background Technology

[0002] Brucellosis (hereinafter referred to as Brucella) is a widespread zoonotic disease caused by Brucella bacteria. Currently, Brucella is classified into 12 species based on host preference and traditional biochemical characteristics. Different species of Brucella have characteristic host preferences, but cross-species transmission frequently occurs, complicating the diagnosis and epidemiological control of brucellosis. Recent surveys indicate that there are 1.6 million to 2.1 million new human cases of brucellosis worldwide each year, significantly exceeding the previous estimate of 500,000. Although most human brucellosis infections originate from direct contact with infected livestock or consumption of contaminated dairy products, pets such as dogs and cats are increasingly considered important hosts for brucellosis infection.

[0003] Brucella canis is a naturally occurring, rough-type bacterium that primarily infects dogs, causing reproductive disorders and potentially leading to chronic, asymptomatic infection, thus complicating diagnosis. Furthermore, rough-type antigen testing is typically not performed in human brucellosis diagnosis, making it easy to miss or misdiagnose a pet owner infected with Brucella canis during veterinary testing. Therefore, testing pets for Brucella canis infection is crucial for both the pet and its owner.

[0004] This invention aims to develop a new rapid identification method for canine brucellosis, thereby enabling rapid detection of canine brucellosis and providing a scientific approach for its prevention and control. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid identification method for canine Brucella to address the problems existing in the prior art. This rapid identification method has the advantages of high specificity, high sensitivity, and good reproducibility. It can be directly applied to the screening of pet clinical samples, is simple to operate, and requires no complex equipment. It provides an efficient tool for the rapid identification of canine Brucella and has important practical value for the epidemiological control of brucellosis and the prevention of zoonotic diseases.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a primer and probe combination for detecting Brucella canis, including universal Brucella detection primers and universal Brucella detection probes. wbdA Gene amplification primers, canine Brucella detection probes, and other Brucella detection probes;

[0008] The universal primers for Brucella detection include the upstream primer Brucella-F with the nucleotide sequence shown in SEQ ID NO.1 and the upstream primer Brucella-R with the nucleotide sequence shown in SEQ ID NO.2;

[0009] The wbdA The gene amplification primers include upstream primer wbdA-F with nucleotide sequences as shown in SEQ ID NO.4 and upstream primer wbdA-R with nucleotide sequences as shown in SEQ ID NO.5;

[0010] The nucleotide sequences of the universal Brucella detection probe, the canine Brucella detection probe, and the other Brucella detection probes are shown in SEQ ID NO.3, SEQ ID NO.6, and SEQ ID NO.7, respectively.

[0011] Furthermore, the universal Brucella detection probe, the canine Brucella detection probe, and the other Brucella detection probes are labeled with different fluorescent reporter groups.

[0012] Furthermore, the 5' ends of the universal Brucella detection probe, the canine Brucella detection probe, and the other Brucella detection probes are respectively labeled with ROX fluorescent reporter group, FAM fluorescent reporter group, and VIC fluorescent reporter group.

[0013] Furthermore, the 3' end of the Brucella universal detection probe is labeled with a BHQ-2 fluorescent quencher group;

[0014] The 3' end of both the canine Brucella detection probe and the other Brucella detection probes is labeled with an MGB fluorescent quencher group.

[0015] The present invention also provides the application of the above-described primer and probe combination in the preparation of detection products for Brucella canis.

[0016] Furthermore, the testing product is a reagent kit.

[0017] The present invention also provides a detection product for canine brucellosis, comprising the above-described primer and probe combination.

[0018] Furthermore, the testing product is a reagent kit.

[0019] This invention also provides a rapid identification method for canine brucellosis for non-disease diagnosis or treatment purposes, comprising the following steps:

[0020] Genomic DNA was extracted from the sample to be tested;

[0021] Using the genomic DNA as a template, fluorescence quantitative detection is carried out by using the above primer and probe combination, the signals of the fluorescence channels are collected, and the judgment is made according to the following criteria:

[0022] Establishment conditions: On the premise that the ROX detection channel is positive, it is judged whether Brucella is contained in the test sample; if the ROX detection channel is negative, and any other detection channel is positive, it cannot be judged that Brucella is contained in the test sample;

[0023] Brucella canis: On the premise that the FAM detection channel is positive, when the Ct value of the FAM detection channel < the Ct value of the VIC detection channel or the VIC detection channel is negative, the test sample contains Brucella canis;

[0024] Non - Brucella canis: On the premise that the VIC detection channel is positive, when the Ct value of the VIC detection channel < the Ct value of the FAM detection channel or the FAM detection channel is negative, the test sample contains non - Brucella canis;

[0025] The judgment criteria for negative and positive of the detection channels are as follows:

[0026] Positive: The Ct value of the detection channel ≤ 36, and there is an obvious amplification curve;

[0027] Suspicious: The Ct value of the detection channel is in the range of 36 - 38; at this time, the sample should be retested. If the Ct value of the repeated experiment result is still in the range of 36 - 38 and there is an obvious amplification curve, it is judged as positive, otherwise it is negative;

[0028] Negative: The Ct value of the detection channel > 38 or there is no Ct value.

[0029] Furthermore, the detection system for the fluorescence quantitative detection is: 10 μL of 2×SuperFastStar ProbeMixture, 5.2 μL of sterile and enzyme - free water, 0.4 μL each of the primer and the probe, and 2 μL of the template;

[0030] The reaction program for the fluorescence quantitative detection is: pre - denaturation at 95℃ for 30 s; 95℃ for 10 s, 60℃ for 35 s, 40 cycles, and the corresponding fluorescence signals are collected at the end of each cycle.

[0031] The present invention discloses the following technical effects:

[0032] The present invention screens Brucella canis wbdAA multi-channel quantitative PCR detection system was constructed by designing a proprietary primer and probe combination for gene-specific SNP sites. This primer and probe combination includes universal and species-specific primers and probes for Brucella, enabling simultaneous detection through different fluorescent labels. This allows for rapid confirmation of Brucella infection and accurate differentiation between canine Brucella and other species, effectively addressing the issues of missed diagnoses and misdiagnoses in traditional methods. Specificity experiments showed no cross-reactivity with non-Brucella species and achieved 100% specificity for canine Brucella; the sensitivity reached 2 × 10⁻⁶. 2 The concentration of CFU / μL meets the needs of low-concentration clinical sample detection; the coefficient of variation of Ct values ​​in repeatability experiments is small, and the results are stable and reliable. The detection method and corresponding detection products provided by this invention can be directly applied to the screening of pet clinical samples. The operation is simple and does not require complex equipment, providing an efficient tool for the rapid identification of Brucella in canine breeds. It has important practical value for the epidemiological control of brucellosis and the prevention of zoonotic diseases. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a sequence alignment diagram of the wbdA gene from different Brucella species.

[0035] Figure 2 This is a diagram showing the results of detecting Brucella canis using probe wbdA-probe-1 in Example 1;

[0036] Figure 3 This is a diagram showing the results of detecting Brucella mesenteriae using probe wbdA-probe-2 in Example 1;

[0037] Figure 4 This is a graph showing the specific detection results in Example 1;

[0038] Figure 5 This is a graph showing the sensitivity detection results in Example 1;

[0039] Figure 6 The image shows the detection results of the clinical samples in Example 2; where A represents the detection results of the universal detection probe for Brucella species in the clinical samples; B represents the detection results of Brucella canis; and C represents the detection results of Brucella species other than those in the clinical samples. Detailed Implementation

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0043] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0044] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0045] The experimental materials involved in the following examples are as follows:

[0046] (1) Strains and culture

[0047] The Brucella species involved in this invention, including those from sheep, pigs, cattle, dogs, and sheep epididymis, were preserved, passaged, cultured, counted, and inactivated by the Institute of Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention. The Escherichia coli ATCC25922, Salmonella pullorum CVCC526, Salmonella typhimurium ATCC14028, Listeria monocytogenes ATCC 19115, Bacillus anthracis (vaccine strain), Mycobacterium tuberculosis (vaccine strain), Pasteurella multocida (isolated strain), Bacillus cereus (isolated strain), and Aureobacterium spp. (isolated strain) were all preserved, passaged, cultured, counted, and inactivated by the Animal Biosafety and Public Health Control Team of the Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0048] Clinical samples were prepared by the Animal Biosafety and Public Health Prevention and Control Team of the Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences, in accordance with the "Diagnostic Techniques for Animal Brucellosis" (GB / T 18646-2025) and "Real-time Fluorescent PCR Detection of Brucella in Animals" (T / CVMA 20-2020).

[0049] The genomes, genes, and encoded protein sequences of different Brucella standard strains were downloaded from the NCBI RefSeq database (ftp: / / ftp.ncbi.nlm.nih.gov / genomes / ) for gene alignment and screening.

[0050] (2) Main reagents and instruments

[0051] The Wizard Genomic DNA Purification Kit was purchased from Prometheus (Beijing) Biotechnology Co., Ltd., and the DNeasy Blood & Tissue Kit was purchased from Qiager Biotechnology Co., Ltd. Primers and probes for quantitative fluorescence detection were synthesized by Sangon Biotech (Shanghai) Co., Ltd., sterile enzyme-free water was purchased from Beijing Solarbio Technology Co., Ltd., and 2×SuperFastStar Probe Mixture was purchased from Jiangsu Kangwei Century Co., Ltd.

[0052] The instruments involved in this invention, including NanoDrop One (Thermo Scientific), GENTIER 96 quantitative PCR instrument (Xi'an Tianlong Technology Co., Ltd.), PCR instrument (BioRad), and DensiCHEK Plus electronic turbidimeter (BioMerieux), were all provided by the Animal Biosafety and Public Health Prevention and Control Team of the Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0053] Example 1: Construction of a method for screening and detecting Brucella canis-specific SNP sites

[0054] 1. Experimental Methods

[0055] 1.1 Sequence alignment and genome extraction

[0056] The gene sequence for LPS synthesis in Brucella was extracted and compared to identify canine Brucella-specific sequences and SNPs. The genome was extracted and purified according to the Wizard Genomic DNA Purification Kit instructions, and its concentration was detected using NanoDrop One. After appropriate dilution, it was used as a template for subsequent detection.

[0057] Primers were designed based on the 300 bp sequence before and after the screening specific sequences and SNPs, amplified, and sequenced to detect whether the specific sequences and SNPs were present in canine Brucella.

[0058] 1.2 Establishment of MGB probe method

[0059] Universal primers and probes for Brucella detection were designed. Probes with sequences and MGB-labeled SNPs specific to canine Brucella were designed to detect different Brucella species.

[0060] The amplification system consisted of 20 μL / sample, including 10 μL of 2×SuperFastStar Probe Mixture, 5.2 μL of sterile enzyme-free water, 0.4 μL each of primers and probes (4 primers and 3 probes, each with a final concentration of 0.2 μM), and 2 μL of template. A two-step amplification and detection method was used, with the reaction program as follows: pre-denaturation at 95℃ for 30 s; 95℃ for 10 s, 60℃ for 35 s, for 40 cycles. The corresponding fluorescence signal was collected at the end of each cycle.

[0061] 1.3 Specificity Detection

[0062] The method established in this invention is used to detect other bacteria of different species, with a positive control of 2 × 10⁻⁶. 5 CFU (Chronic Fusarium oxysporum) of canine brucellosis, with a negative control of enzyme-free sterile water. Other bacteria included: Escherichia coli ATCC 25922, Salmonella pullorum CVCC526, Salmonella typhimurium ATCC14028, Listeria monocytogenes ATCC 19115, Bacillus anthracis (Sterne), Mycobacterium tuberculosis, Pasteurella multocida, Bacillus cereus, and Aureobacterium spp.

[0063] 1.4 Sensitivity Detection

[0064] The quantitatively inactivated drug-resistant bacterial solution was diluted to 1×10⁻⁶. 4 -10 0 CFU / μL was detected using the method established in this invention.

[0065] 1.5 Repeatability Test

[0066] Dilute the quantitatively inactivated Brucella canis bacterial suspension to 1×10⁻⁶. 5 CFU / μL, 2 μL were taken within three days and tested according to the method established in this invention to perform repeatability testing experiments.

[0067] 2. Experimental Results

[0068] 2.1 Specific SNP sites exist in canine Brucella.

[0069] Thirty-one gene sequences reported to be involved in Brucella LPS synthesis were extracted and compared among different Brucella species. It was found that canine and swine species had high sequence similarity, but canine Brucella-specific SNPs were found in the per, lpxE, and wbdA genes.

[0070] Primers were designed to target the specific SNPs of the above three genes for PCR amplification and sequencing verification. It was found that the specific sites of the three genes of Brucella canis were present, and the bases at these sites were the same in other Brucella species. Figure 1 These three gene-specific SNP sites serve as candidate SNP sites for the subsequent development of detection methods.

[0071] 2.2 Successfully established the MGB probe method for detecting Brucella canis.

[0072] Primer pairs and probe pairs were designed for the breed-specific SNP sites of Brucella canis in the above three genes, and different Brucella species were detected. The results showed that only the breed-specific SNP site (503 C / T) of the wbdA gene could effectively distinguish between canine and other Brucella species. The primers and probes are shown in Table 1.

[0073] Table 1 Primer and probe sequences in this invention

[0074]

[0075] After mixing the primer pairs and probe pairs in Table 1, Brucella species from dogs and sheep were detected separately. The bacterial suspensions were 2 × 10⁻⁶ for both species. 5 CFU, with enzyme-free water as the blank control. Quantitative fluorescence amplification revealed that the Ct values ​​for Brucella-probe in detecting canine and sheep Brucella were 22.941 and 23.090, respectively, while the Ct value for wbdA-probe-1 in detecting canine Brucella was 21.762. Figure 2 The Ct value of Brucella mesenteriae detected by probe wbdA-probe-2 was 21.848. Figure 3 ).

[0076] The above results show that the different primer pairs and probe pairs used in this invention do not interfere with each other.

[0077] 2.3 Specific detection results

[0078] The method established in this invention is used to detect other bacteria of different species, with a positive control of 2 × 10⁻⁶. 5 CFU of *Brucella canis* was used, with enzyme-free sterile water as the negative control. After amplification, the Ct value of the FAM probe for detecting *Brucella canis* was 22.051, while no amplification curve was observed for other bacterial species, indicating that the method established in this invention has good specificity. Figure 4 ).

[0079] 2.4 Sensitivity test results

[0080] The quantitatively inactivated drug-resistant bacterial solution was diluted to 1×10⁻⁶. 4 -10 0 CFU / μL was detected using the primer and probe pairs in Table 1. The primer and probe pairs for *Brucella canis* showed a detection rate of 2 × 10⁻⁶. 2 The Ct value for CFU / μL bacterial amplification was 34.551, 2×10⁻⁶. 1 The CFU (Cat value) for Brucella canis was 38.160. These results indicate that the *Brucella canis* strain established in this invention can detect 2 × 10⁻⁶ bacteria. 2 CFU / μL bacterial count ( Figure 5 ).

[0081] 2.5 Repeatability test results

[0082] Quantitatively inactivated Brucella canis bacterial suspension diluted to 1×10 5 CFU / μL, 2 μL were taken within three days and tested according to the method established in this invention. The Ct values ​​were found to be 22.132, 21.956 and 22.243 (mean ± standard deviation = 22.110 ± 0.145), respectively, showing good repeatability.

[0083] Example 2: Nucleic acid extraction and detection from clinical samples

[0084] A total of 302 dog / cat samples suspected of having brucellosis were collected from pet hospitals across the country. Seven positive samples were identified through rose benzene plate agglutination and microagglutination tests. Following the instructions of the DNeasy Blood & Tissue Kit, nucleic acid was extracted from the clinically positive brucellosis samples. After concentration testing, 2 μL of the sample was taken and tested using the method established in Example 1. Simultaneously, a positive control (canine brucellosis) and a negative control (enzyme-free sterile water) were set up.

[0085] Collect signals from the fluorescence channel and judge them according to the following criteria:

[0086] Establishment conditions: Determine whether Brucella is present in the test sample on the premise that the ROX detection channel is positive; if the ROX detection channel is negative, and any other detection channel is positive, it cannot be determined that Brucella is present in the test sample.

[0087] Brucella canis: On the premise that the FAM detection channel is positive, when the Ct value of the FAM detection channel < the Ct value of the VIC detection channel or the VIC detection channel is negative, then the test sample contains Brucella canis.

[0088] Non - Brucella canis: On the premise that the VIC detection channel is positive, when the Ct value of the VIC detection channel < the Ct value of the FAM detection channel or the FAM detection channel is negative, then the test sample contains non - Brucella canis.

[0089] The judgment criteria for negative and positive of the detection channels are as follows:

[0090] Positive: The Ct value of the detection channel ≤ 36, with an obvious amplification curve.

[0091] Suspicious: The Ct value of the detection channel is in the range of 36 - 38; at this time, the sample should be retested. If the Ct value of the repeated experiment result is still in the range of 36 - 38 and there is an obvious amplification curve, it is determined as positive, otherwise it is negative.

[0092] Negative: The Ct value of the detection channel > 38 or there is no Ct value.

[0093] The detection results of clinical samples are shown in Figure 6 , and the results show that 5 nucleic acid detection results of Brucella are positive ( Figure 6 A in Figure 6 ), among which 1 is positive for Brucella canis ( Figure 6 B in

[0094] The above - described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer and probe combination for multi-channel quantitative fluorescence detection of Brucella canis, characterized in that, Including universal primers for Brucella detection, universal probes for Brucella detection, wbdA Gene amplification primers, canine Brucella detection probes, and other Brucella detection probes; The universal detection primers for Brucella include an upstream primer Brucella-F with a nucleotide sequence as shown in SEQ ID NO.1 and an upstream primer Brucella-R with a nucleotide sequence as shown in SEQ ID NO.2; The wbdA The gene amplification primers include upstream primer wbdA-F with nucleotide sequences as shown in SEQ ID NO.4 and upstream primer wbdA-R with nucleotide sequences as shown in SEQ ID NO.5; The nucleotide sequences of the universal detection probe for Brucella, the detection probe for Brucella canis, and the detection probe for other species of Brucella are as shown in SEQ ID NO.3, SEQ ID NO.6, and SEQ ID NO.7 respectively; The 5' ends of the universal detection probe for Brucella, the detection probe for Brucella canis, and the detection probe for other species of Brucella are respectively labeled with ROX fluorescent reporter group, FAM fluorescent reporter group, and VIC fluorescent reporter group; The 3' end of the universal detection probe for Brucella is labeled with BHQ-2 fluorescent quenching group; The 3' ends of the detection probe for Brucella canis and the detection probe for other species of Brucella are both labeled with MGB fluorescent quenching group.

2. Use of the primer and probe combination according to claim 1 in the preparation of a detection product for Brucella canis.

3. The application according to claim 2, characterized in that, The detection product is a kit.

4. A detection product for canine brucellosis, characterized in that, It includes the primer and probe combination according to claim 1.

5. The testing product according to claim 4, characterized in that, The detection product is a kit.

6. A rapid identification method for canine brucellosis for non-disease diagnosis or treatment purposes, characterized in that, It includes the following steps: Genomic DNA of the test sample is extracted; Using the genomic DNA as a template, fluorescence quantitative detection is carried out with the primer and probe combination according to claim 1, the signals of the fluorescence channels are collected, and the judgment is made according to the following criteria: Establishment condition: Judge whether Brucella is contained in the test sample on the premise that the ROX detection channel is positive; If the ROX detection channel is negative, and any other detection channel is positive, it cannot be judged that Brucella is contained in the test sample; Brucella canis: On the premise that the FAM detection channel is positive, when the Ct value of the FAM detection channel < the Ct value of the VIC detection channel or the VIC detection channel is negative, then the test sample contains Brucella canis; Non-Brucella canis: On the premise that the VIC detection channel is positive, when the Ct value of the VIC detection channel < the Ct value of the FAM detection channel or the FAM detection channel is negative, then the test sample contains non-Brucella canis; The judgment criteria for negative and positive of the detection channels are as follows: Positive: The Ct value of the detection channel ≤ 36, and there is an obvious amplification curve; Suspicious: The Ct value of the detection channel is in the range of 36 - 38; At this time, the sample should be retested. If the Ct value of the repeated experiment result is still in the range of 36 - 38 and there is an obvious amplification curve, it is judged as positive, otherwise it is negative; Negative: The Ct value of the detection channel > 38 or there is no Ct value.

7. The rapid identification method according to claim 6, characterized in that, The detection system for the fluorescence quantitative detection is: 10 μL of 2×SuperFastStar Probe Mixture, 5.2 μL of sterile and enzyme-free water, 0.4 μL of each primer and probe, and 2 μL of template; The reaction program for the fluorescence quantitative detection is: pre-denaturation at 95℃ for 30 s; at 95℃ for 10 s, at 60℃ for 35 s, 40 cycles, and the corresponding fluorescence signals are collected at the end of each cycle.

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