Rapid identification method of Brucella canis

Abstract

The invention discloses a rapid identification method of Brucella canis, and relates to the technical field of molecular detection. The rapid identification method comprises the following steps: extracting genome DNA of a sample to be detected; the genome DNA is used as a template, a primer and probe combination is used for fluorescence quantitative detection, signals of a fluorescence channel are collected, and whether a sample to be detected is Brucella canis or not is identified. According to the invention, a multi-channel fluorescent quantitative detection system is constructed by screening specific SNP (Single Nucleotide Polymorphism) sites of a wbdA gene of Brucella canis and designing a specific primer and probe combination. The primer and probe combination comprises a Brucella universal primer probe and a species specific primer probe, synchronous detection is achieved through different fluorescence labels, Brucella infection can be rapidly confirmed, dog species and other species of Brucella can be accurately distinguished, an efficient tool is provided for rapid identification of the dog species of Brucella, and the primer and probe combination is suitable for rapid identification of the dog species of Brucella. The method has an important practical value for the epidemiological prevention and control of brucellosis and the prevention of zoonosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular detection, in particular to a rapid identification method of canine Brucella. BACKGROUND

[0002] Brucellosis (hereinafter referred to as brucellosis) is a widespread zoonosis caused by Brucella. At present, Brucella is divided into 12 species according to host preference and traditional biochemical characteristics. Different species of Brucella have characteristic host preferences, but cross-species transmission often occurs, which complicates the diagnosis and epidemiological prevention and control of brucellosis. Recent survey results show that there are 1.6-2.1 million new human brucellosis cases worldwide each year, which is much higher than the previous estimate of 0.5 million cases. Although most human brucellosis infections are caused by direct contact with infected livestock or consumption of contaminated dairy products, pets such as dogs and cats are increasingly recognized as important infection hosts of brucellosis.

[0003] Canine Brucella is a natural rough type, mainly infecting dogs and causing reproductive disorders, and can also cause chronic asymptomatic infections, making diagnosis more complicated. Moreover, rough type antigen detection is usually not performed during human brucellosis diagnosis, so if a pet owner is infected with canine Brucella, it is easy to miss or misdiagnose during hospital detection. Therefore, detecting whether pets are infected with canine Brucella is very important for both pets and pet owners.

[0004] The present application aims to develop a new rapid identification method for canine Brucella to achieve rapid detection of canine Brucella and provide a scientific method for the prevention and control of brucellosis. SUMMARY

[0005] The purpose of the present application is to provide a rapid identification method for canine Brucella to solve the problems existing in the prior art. The rapid identification method has the advantages of high specificity, high sensitivity and good repeatability, can be directly applied to pet clinical sample screening, is simple to operate and does not require complex equipment, provides an efficient tool for rapid identification of canine Brucella, and has important practical value for the epidemiological prevention and control of brucellosis and the prevention of zoonosis.

[0006] To achieve the above purpose, the present application provides the following solutions: The present application provides a primer and probe combination for detecting canine Brucella, which comprises a Brucella universal detection primer, a Brucella universal detection probe, wbdA a gene amplification primer, a canine Brucella detection probe and an other-species Brucella detection probe; The Brucella universal detection primer comprises 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 Brucella universal detection primer comprises 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. wbdA The Brucella universal detection primer comprises 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 nucleotide sequences of the Brucella universal detection probe, the canine Brucella detection probe and the other-species Brucella detection probe are shown in SEQ ID NO. 3, SEQ ID NO. 6 and SEQ ID NO. 7, respectively.

[0007] Further, the Brucella universal detection probe, the canine Brucella detection probe and the other-species Brucella detection probe are labeled with different fluorescent reporter groups.

[0008] Further, the 5' ends of the Brucella universal detection probe, the canine Brucella detection probe and the other-species Brucella detection probe are labeled with ROX fluorescent reporter group, FAM fluorescent reporter group and VIC fluorescent reporter group, respectively.

[0009] Further, the 3' end of the Brucella universal detection probe is labeled with BHQ-2 fluorescent quenching group; The 3' ends of the canine Brucella detection probe and the other-species Brucella detection probe are both labeled with MGB fluorescent quenching group.

[0010] The application also provides the use of the above-mentioned primer and probe combination in the preparation of a detection product for canine Brucella.

[0011] Further, the detection product is a kit.

[0012] The application also provides a detection product for canine Brucella, comprising the above-mentioned primer and probe combination.

[0013] Further, the detection product is a kit.

[0014] The application also provides a rapid identification method for canine Brucella for non-disease diagnosis or treatment purposes, comprising the following steps: Extracting the genomic DNA of the sample to be detected; Using the above-mentioned primer and probe combination for fluorescent quantitative detection with the genomic DNA as a template, collecting the signals of the fluorescence channels, and judging according to the following standards: Establishment condition: judge whether the sample contains Brucella under 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 the sample contains Brucella; Canine Brucella: under the premise that the FAM detection channel is positive, when the FAM detection channel Ct value < VIC detection channel Ct value or the VIC detection channel is negative, the sample contains canine Brucella; Non-canine Brucella: under the premise that the VIC detection channel is positive, when the VIC detection channel Ct value < FAM detection channel Ct value or the FAM detection channel is negative, the sample contains non-canine Brucella; The judgment criteria of the negative and positive of the detection channel are as follows: Positive: the detection channel Ct value <= 36, and there is an obvious amplification curve; Suspicious: the detection channel Ct value is in the range of 36-38; at this time, the sample should be repeatedly detected, if the repeated experimental result Ct value 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 detection channel Ct value > 38 or there is no Ct value.

[0015] Further, the detection system of the fluorescent quantitative detection is: 10 muL 2xSuperFastStar Probe Mixture, 5.2 muL sterile enzyme-free water, 0.4 muL of primers and probes respectively and 2 muL of template; The reaction procedure of the fluorescent quantitative detection is: pre-denaturation 95 DEG C for 30 s; 95 DEG C for 10 s, 60 DEG C for 35 s, 40 cycles, and the corresponding fluorescence signal is collected at the end of each cycle.

[0016] The present application discloses the following technical effects: The present application screens canine Brucella wbdA The present application screens canine Brucella The primer and probe combination contains Brucella universal primer probe and species-specific primer probe, and realizes synchronous detection through different fluorescent labels, which can quickly confirm Brucella infection, accurately distinguish canine and other species Brucella, and effectively solve the problems of missed diagnosis and misdiagnosis in traditional detection. The specificity experiment shows that the method has no cross reaction to non-Brucella, and the detection specificity to canine Brucella reaches 100%; the sensitivity can reach 2x10 2The CFU / ul meets the detection requirement of a clinical low-concentration sample; and the Ct value coefficient of variation in a repeated experiment is small, and the result is stable and reliable. The detection method and the corresponding detection product provided by the application can be directly applied to pet clinical sample screening, and the operation is simple and does not require complex equipment, thereby providing an efficient tool for rapid identification of canine Brucella, and having important practical value for the epidemiological prevention and control of Brucellosis and the prevention of zoonosis. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0018] Figure 1 Sequence alignment chart of wbdA genes of different Brucella species; Figure 2 Result chart of detecting canine Brucella by the probe wbdA-probe-1 in Example 1; Figure 3 Result chart of detecting ovine Brucella by the probe wbdA-probe-2 in Example 1; Figure 4 Specificity detection result chart in Example 1; Figure 5 Sensitivity detection result chart in Example 1; Figure 6 Detection result chart of clinical samples in Example 2; wherein, A is the detection result of a clinical sample Brucella genus and species universal detection probe; B is the detection result of canine Brucella; and C is the detection result of other Brucella species. DETAILED DESCRIPTION

[0019] The various exemplary embodiments of the present application will now be described in detail below, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0020] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value in the stated range, and any other stated value or intermediate value in the stated range, is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0021] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, unless indicated otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described therein. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0022] Many modifications and variations of this application of the application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0023] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed materials and methods.

[0024] The experimental materials involved in the following examples are as follows: (1) Strains and culture The ovine species, porcine species, bovine species, canine species, and ovine epididymal species Brucella involved in the present application are preserved, passaged, cultured, counted, and inactivated by the Center for Disease Control and Prevention of Infectious Diseases, China. The E. coli ATCC25922, chicken pullorum Salmonella CVCC526, murine typhus Salmonella ATCC14028, Listeria monocytogenes ATCC 19115, Bacillus anthracis Sterne (vaccine strain), Mycobacterium tuberculosis (vaccine strain), Pasteurella multocida (isolated strain), Pseudomonas aeruginosa (isolated strain), and Chryseobacterium (isolated strain) involved in the specific detection are preserved, passaged, cultured, counted, and inactivated by the Animal Biosecurity and Public Health Prevention and Control Team of the Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0025] The clinical samples are completed by the Animal Biosecurity and Public Health Prevention and Control Team of the Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences according to the "Animal Brucella Diagnosis Technology" (GB / T 18646-2025) and "Animal Brucella Real-time Fluorescent PCR Detection" (T / CVMA 20-2020).

[0026] The genomes, genes, and coding protein sequences of different species of Brucella standard strains are downloaded from the NCBI RefSeq database (ftp: / / ftp.ncbi.nlm.nih.gov / genomes / ) for gene alignment and screening.

[0027] (2) Main reagents and instruments The Wizard Genomic DNA Purification Kit was purchased from Promega (Beijing) Biotechnology Co., Ltd., the nucleic acid extraction kit DNeasy Blood & Tissue Kit was purchased from Kajia Biotechnology Co., Ltd.; the primers and probes used in the fluorescent quantitative detection were synthesized by Shenguo Biotechnology (Shanghai) Co., Ltd., sterile enzyme-free water was purchased from Beijing Solabio Technology Co., Ltd., and 2xSuperFastStar Probe Mixture was purchased from Jiangsu Kangwei Century Co., Ltd.

[0028] The instruments involved in the present application, NanoDrop One (Thermo Scientific), fluorescent quantitative PCR instrument GENTIER 96 (Xi'an Tianlong Technology Co., Ltd.), PCR instrument (BioRad), and electronic turbidimeter DensiCHEK Plus (BioMerieux), were provided by the Animal Biosafety and Public Health Prevention Team of the Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0029] Example 1: Screening and detection method for canine Brucella specific SNP site 1. Experimental method 1.1 Sequence alignment and genome extraction The gene sequence of Brucella for synthesizing LPS was extracted and then aligned to screen for sequences and SNPs sites specific to canine Brucella. The genome was extracted and purified according to the Wizard Genomic DNA Purification Kit operation manual, the concentration was detected by NanoDrop One, and the template was appropriately diluted for subsequent detection.

[0030] Primers were designed for the 300 bp sequences before and after the specific sequences and SNPs, amplified and sequenced to detect whether the specific sequences and SNPs exist in canine Brucella.

[0031] 1.2 MGB probe method establishment Universal detection primers and probes for Brucella were set. MGB-labeled probes specific to the sequences and SNPs of canine Brucella were designed to detect different species of Brucella.

[0032] 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.

[0033] 1.3 Specificity Detection 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.

[0034] 1.4 Sensitivity Detection 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.

[0035] 1.5 Repeatability Test 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.

[0036] 2. Experimental Results 2.1 Specific SNP sites exist in canine Brucella. 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.

[0037] 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.

[0038] 2.2 Successful establishment of MGB probe method for detecting canine Brucella The primer pair and probe pair for the above-mentioned three genes of canine Brucella specific SNP sites were designed, and different species of Brucella were detected. It was found that only the canine specific SNP site (503 C / T) of the wbdA gene could effectively distinguish canine Brucella from other species of Brucella. The primers and probes are shown in Table 1.

[0039] Table 1 Primer and probe sequences in the application

[0040] The primer pair and probe pair in Table 1 were mixed and used to detect canine and sheep Brucella, and the bacterial solution was 2×10 5 CFU, and the blank control was enzyme-free water. After fluorescence quantitative amplification, it was found that the Ct values of the probes Brucella-probe for detecting canine and sheep Brucella were 22.941 and 23.090, respectively, the Ct value of the probe wbdA-probe-1 for detecting canine Brucella was 21.762 ( Figure 2 ), and the Ct value of the probe wbdA-probe-2 for detecting sheep Brucella was 21.848 ( Figure 3 ).

[0041] The above results show that the different primer pairs and probe pairs used in the application do not interfere with each other.

[0042] 2.3 Specific detection results The method established in the application was used to detect different species of other bacteria, and the positive control was 2×10 5 CFU of canine Brucella, and the negative control was enzyme-free sterile water. After amplification, it was found that the Ct value of the FAM probe for detecting canine Brucella was 22.051, and there was no amplification curve for detecting other species of bacteria, indicating that the method established in the application has good specificity ( Figure 4 ).

[0043] 2.4 Sensitivity detection results The quantitatively inactivated drug-resistant bacteria were diluted to 1×10 4 -10 0 CFU / μL, and the primer pair and probe pair in Table 1 were used for detection. It was found that the Ct value of the primer and probe for detecting 2×10 2 CFU / μL bacteria was 34.551, and the Ct value for detecting 2×10 1 CFU bacteria was 38.160. The above results show that the canine Brucella established in the application can detect 2×10 2 CFU / μL of bacteria ( Figure 5 ).

[0044] 2.5 repeatability test results The inactivated canine Brucella bacteria solution was diluted to 1 x 10 5 CFU / μL, 2 μL of each was taken for detection according to the method established in the present application within three days, and it was found that the Ct values were 22.132, 21.956 and 22.243 (average value ± standard deviation = 22.110 ± 0.145) respectively, and the repeatability was good.

[0045] Example 2: Extraction and detection of nucleic acid of clinical samples A total of 302 samples of suspected Brucella disease from pet hospitals all over the country were collected, and 7 positive samples were found by the Rose Bengal plate agglutination test and micro agglutination test. According to the operation instruction of DNeasy Blood & Tissue Kit, the nucleic acid of the Brucella disease clinical samples verified as positive was extracted, 2 μL of the sample was taken after detection of the concentration, and the method established in Example 1 was used for detection. At the same time, a positive control (canine Brucella) and a negative control (enzyme-free sterile water) were set.

[0046] The signals of the fluorescence channels were collected, and the judgment was made according to the following standards: Established condition: judge whether the Brucella bacteria are contained in the sample to be tested under the premise that the ROX detection channel is positive; if the ROX detection channel is negative and any of the other detection channels is positive, it cannot be judged that the sample to be tested contains Brucella bacteria; Canine Brucella: under the premise that the FAM detection channel is positive, when the FAM detection channel Ct value < VIC detection channel Ct value or the VIC detection channel is negative, the sample to be tested contains canine Brucella; Non-canine Brucella: under the premise that the VIC detection channel is positive, when the VIC detection channel Ct value < FAM detection channel Ct value or the FAM detection channel is negative, the sample to be tested contains non-canine Brucella; The judgment standards of the negative and positive of the detection channels are as follows: Positive: the Ct value of the detection channel is ≤ 36, and there is a clear amplification curve; Suspected: the Ct value of the detection channel is in the range of 36-38; at this time, the sample should be repeatedly detected, and if the Ct value of the repeated experiment is still in the range of 36-38 and there is a clear amplification curve, it is determined to be positive, otherwise it is negative; Negative: the Ct value of the detection channel is > 38 or there is no Ct value.

[0047] The detection results of the clinical samples are shown in Table Figure 6 , and the results show that 5 samples of Brucella nucleic acid detection results are positive ( Figure 6 , among which 1 sample is positive for canine Brucella ( Figure 6 , and 4 samples are positive for other species of Brucella nucleic acid ( Figure 6C).

[0048] The above-described embodiments are merely intended to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements of the present application made by those skilled in the art based on the above-described embodiments should fall within the scope of the present application defined by the claims.

Claims

1. A primer and probe combination for detecting canine Brucella spp. 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 primer of Brucella comprises 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 primer includes an upstream primer wbdA-F with a nucleotide sequence as shown in SEQ ID NO. 4 and an upstream primer wbdA-R with a nucleotide sequence as shown in SEQ ID NO. 5; The nucleotide sequences of the universal detection probe of Brucella, the detection probe of B. canis and the detection probe of other Brucella species are shown in SEQ ID NO. 3, SEQ ID NO. 6 and SEQ ID NO. 7 respectively.

2. The primer and probe combination of claim 1, wherein, The universal detection probe of Brucella, the detection probe of B. canis and the detection probe of other Brucella species are labeled with different fluorescent reporter groups.

3. The primer and probe combination of claim 2, wherein, The 5' ends of the universal detection probe of Brucella, the detection probe of B. canis and the detection probe of other Brucella species are labeled with ROX fluorescent reporter group, FAM fluorescent reporter group and VIC fluorescent reporter group respectively.

4. The primer and probe combination of claim 3, wherein, The 3' end of the universal detection probe of Brucella is labeled with BHQ-2 fluorescent quenching group; The 3' ends of the detection probe of B. canis and the detection probe of other Brucella species are both labeled with MGB fluorescent quenching group.

5. Use of the primer and probe combination of any one of claims 1-4 in the preparation of a detection product of B. canis.

6. Use according to claim 5, characterized in that, The detection product is a kit.

7. A product for detecting canine Brucella species, characterized by, The detection product comprises the primer and probe combination of any one of claims 1-4.

8. The test product of claim 5, wherein, The detection product is a kit.

9. A method for rapid identification of canine Brucella species for non-diagnostic or therapeutic purposes, characterized by, The method comprises the following steps: Extracting genomic DNA from the sample to be detected; Using the primer and probe combination of any one of claims 3-4 for fluorescent quantitative detection with the genomic DNA as a template, collecting the signals of the fluorescent channels and judging according to the following criteria: Established condition: judging whether the sample to be detected contains Brucella under the premise that the ROX detection channel is positive; If the ROX detection channel is negative and any of the other detection channels is positive, it cannot be judged that the sample to be detected contains Brucella; B. canis: under the premise that the FAM detection channel is positive, when the FAM detection channel Ct value < the VIC detection channel Ct value or the VIC detection channel is negative, the sample to be detected contains B. canis; Non-B. canis: under the premise that the VIC detection channel is positive, when the VIC detection channel Ct value < the FAM detection channel Ct value or the FAM detection channel is negative, the sample to be detected contains non-B. canis; The judgment criteria for the negativity and positivity of the detection channels are as follows: Positive: the detection channel Ct value ≤ 36, with obvious amplification curve; Suspected: the detection channel Ct value is in the range of 36-38; at this time, the sample should be repeatedly detected, if the Ct value of the repeated experiment is still in the range of 36-38 and there is obvious amplification curve, it is judged as positive, otherwise as negative; Negative: the detection channel Ct value > 38 or without Ct value.

10. The rapid identification method according to claim 9, characterized in that, The detection system for the fluorescent quantitative detection is: 10 μL 2×SuperFastStar Probe Mixture, 5.2 μL sterile water, 0.4 μL of each of the primer and probe and 2 μL of template. The reaction procedure of the fluorescent quantitative detection is: pre-denaturation at 95℃ for 30 s; 95℃ for 10 s, 60℃ for 35 s, 40 cycles, and the corresponding fluorescent signal is collected at the end of each cycle.

Images