Primer probe group, kit, method and application for detecting brucella

Abstract

The invention discloses a primer probe group, a kit, a method and application for detecting brucella. Belongs to the technical field of animal epidemic disease and zoonosis detection. The important virulence factor T4SS virB10 of Brucella is taken as a target gene, a high-specificity primer probe group is designed, and a high-sensitivity ddPCR detection kit and a detection method are constructed through optimization experiments, so that absolute quantification of Brucella is realized, the detection sensitivity is improved, false negative and false positive risks are reduced, and the kit has a good application prospect. The kit can effectively improve the detection rate of early infection or recessive infection, and can be used as a reference standard for screening and performance evaluation of clinical detection kits.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of animal epidemic and zoonosis detection, in particular to a primer probe set, kit, method and application for detecting Brucella. BACKGROUND

[0002] Brucellosis is a widespread zoonosis, and after infection, animals will have symptoms such as joint pain, miscarriage, infertility and reduced milk production, which seriously threatens animal food and public health safety and causes great economic losses. Sick animals are the main source of human brucellosis, and humans are infected and sickened by contacting sick animals and their products or contaminants. After humans get sick, they will have symptoms such as intermittent fever, arthritis, orchitis and depression. The high positive rate of brucellosis in livestock is the root cause of the gradual increase in the infection rate of human brucellosis, so controlling and purifying Brucella from the source is the key to eradicating brucellosis.

[0003] Brucellosis is mostly non-typical, and is easily misdiagnosed in the early stage of infection. The currently approved Bifidobacterium abortum strains S19 and RB51 vaccines in the global market still have the shortcomings of interfering with clinical diagnosis, causing human disease and leading to miscarriage in pregnant animals, and lack of safe and high-protection vaccines and drugs. Therefore, there is an urgent need for efficient and accurate pathogen detection technology for early monitoring and timely prevention and control of the disease.

[0004] Currently, the main methods for detecting brucellosis etiology are bacterial isolation and culture and molecular biology detection methods. Bacterial isolation and culture is the gold standard for diagnosing Brucella infection, but has the disadvantages of long time consumption (primary culture usually takes 3-30 days) and high risk, and has biological safety risks. Molecular biology detection methods mainly include PCR, fluorescent PCR, CRISPR / Cas gene editing technology, loop-mediated isothermal amplification (LAMP), metagenomic sequencing technology (mNGS), single nucleotide polymorphism (SNP) genotyping technology and microfluidic chip (MC) technology. Among them, metagenomic sequencing technology (mNGS), single nucleotide polymorphism (SNP) genotyping technology and microfluidic chip (MC) technology are high in cost and cannot be applied on a large scale; CRISPR / Cas gene editing technology and loop-mediated isothermal amplification (LAMP) have the disadvantages of low sensitivity and high false positive rate; PCR and fluorescent PCR are widely used in clinical detection of Brucella, but they will have false positive and false negative results when detecting low-abundance samples, and rely on standard curves for nucleic acid quantification.

[0005] Therefore, how to develop a primer probe set, kit, method and application for detecting Brucella, improve the specificity and sensitivity of detection, effectively avoid false positive and false negative risks, and realize absolute quantification of Brucella, is a technical problem that needs to be solved by those skilled in the art. SUMMARY

[0006] Therefore, the application provides a primer probe set, a kit, a method and an application for detecting Brucella, selects a Brucella virulence factor T4SS virB10 as a target sequence, constructs a primer probe set, and a ddPCR detection kit and method, realizes absolute quantification of Brucella, improves the sensitivity and specificity of detection, and effectively avoids the risk of false negative and false positive.

[0007] In order to achieve the above-mentioned purpose, the application adopts the following technical scheme:

[0008] The first purpose of the application is to provide the application of the Brucella virulence factor T4SS virB10 as a target sequence in the detection of Brucella in non-disease diagnosis and treatment.

[0009] Another purpose of the application is to provide a specific primer probe set for detecting Brucella by ddPCR, which comprises a forward primer, a reverse primer and a probe, and the specific sequences are as follows:

[0010] The forward primer is 5'-GGGGTTCAAATCAGATCAACCTC-3', SEQ ID NO.1.

[0011] The reverse primer is 5'-GCGGAATGTTGATCGTATCCTTC-3', SEQ ID NO.2.

[0012] The probe is 5'-CTCGTCGATTCACCTCCGCCGGTA-3', SEQ ID NO.3, the 5' end of the probe is connected with a fluorescent reporter group, and the 3' end is connected with a non-fluorescent quenching group.

[0013] As a preferred technical scheme, the fluorescent reporter group is FAM, and the non-fluorescent quenching group is NFQ-MGB, or other fluorescent reporter groups and non-fluorescent quenching groups.

[0014] Another purpose of the application is to provide the application of the specific primer probe set in the preparation of a kit for detecting Brucella.

[0015] Another purpose of the application is to provide a kit for detecting Brucella by ddPCR, which comprises the specific primer probe set.

[0016] As a preferred technical scheme, the working concentration of the forward primer and the reverse primer is 900 nmol / L, and the working concentration of the probe is 300 nmol / L.

[0017] Another purpose of the application is to provide a method for detecting Brucella by ddPCR, which comprises the following steps:

[0018] (1) extracting DNA of a sample to be tested;

[0019] (2) using the DNA extracted in step (1) as a template, performing ddPCR amplification with the primer probe set.

[0020] As a preferred technical solution, the reaction system of the ddPCR amplification is as follows: 900 nmol / L of the upper and lower primers, 1.8 μL each, 300 nmol / L of the probe, 0.6 μL, ddPCR supermix 10 μL, template 1 μL, and water added to 20 μL.

[0021] As a preferred technical solution, the reaction procedure of the ddPCR amplification is as follows: 95℃ 10min; 94℃ 30s, 60℃ 1min, 40 cycles; 98℃ 10min; 4℃ storage.

[0022] Still another purpose of the present application is to provide the application of the primer probe set, the kit or the method for detecting Brucella by ddPCR in performance evaluation of Brucella commercial kit.

[0023] According to the above technical solution, compared with the prior art, the present application has the following beneficial effects:

[0024] 1) The present application takes the important virulence factor T4SS virB10 of Brucella as a target gene, and designs a high-specificity primer probe set to avoid cross-reaction. Meanwhile, based on the above primer probe set, a high-sensitivity ddPCR detection kit and detection method are constructed through optimization experiments. The method can detect very low copy number of Brucella nucleic acid (1.01 copies / μL), which is significantly better than traditional PCR and qPCR, can effectively reduce the false negative results in clinic, improve the detection rate of early infection or latent infection, realize the absolute quantification of Brucella, overcome the quantitative deviation caused by the difference in amplification efficiency of qPCR, and is especially suitable for accurate quantification and detection of low-abundance samples (such as early infection or trace residual samples).

[0025] 2) In addition, the quantitative results of the ddPCR method of the present application can be used as a reference value to prepare nucleic acid standard products, and as a "gold standard" for screening and performance evaluation (such as minimum detection limit, sensitivity and specificity verification) of clinical detection kits, solving the problem of no unified reference for evaluation of existing kits, helping to standardize the clinical detection market of Brucella, and improving the uniformity of kit quality. BRIEF DESCRIPTION OF DRAWINGS

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 Results of ddPCR primer and probe concentration optimization.

[0028] Figure 2 Results of ddPCR annealing temperature optimization.

[0029] Figure 3 The results are as follows: sensitivity test results of ddPCR method; where A01 is 1.01 × 10⁻⁶. 8 copies / μL; D01 is 1.01×10 7 copies / μL; G01 is 1.01×10 6 copies / μL; B02 is 1.01×10 5 copies / μL; E02 is 1.01×10 4 copies / μL; H02 is 1.01×10 3 copies / μL; CO3 is 1.01×10 2 copies / μL; F03 is 1.01×10 1 copies / μL; A04 is 1.01×10 0 copies / μL.

[0030] Figure 4 For example: ddPCR amplification curve.

[0031] Figure 5 The results are as follows: ddPCR method specificity test results; where C04 is Brucella antigen; A03 is Escherichia coli O157; B03 is Streptococcus suis; C03 is Streptococcus agalactiae; D03 is Salmonella typhimurium; and F03 is Salmonella enteritidis.

[0032] Figure 6 The results of the established Brucella ddPCR method for detecting some clinical samples were presented; B01, C01, E01, F01 and H01 were clinically positive samples, and the rest were clinically negative samples.

[0033] Figure 7 The results of quantitative real-time PCR detection of some clinical samples are shown below. The yellow line represents the fluorescence curve of the positive control, and the rest are the curves of the clinical positive samples. The horizontal axis represents the cycle number, and the vertical axis represents the fluorescence intensity. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Example 1

[0036] Primer and probe design

[0037] Primer and probe sequences were designed based on the gene sequence of T4SS virB10, an important virulence factor of Brucella, in GenBank (accession number: DQ861303.1). The probes and primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the specific sequences are shown in Table 1.

[0038] Table 1 Primer and probe sequences for ddPCR detection of Brucella

[0039]

[0040] Standard plasmid construction

[0041] A standard positive plasmid containing the virB10 target sequence was constructed by Sangon Biotech (Shanghai) Co., Ltd. (using the chemically synthesized and amplified virB10 target sequence as a template, performing conventional PCR, and running the PCR product on agarose gel electrophoresis; the PCR product was recovered using a gel extraction kit, ligated into the PUC57 vector, and the recombinant plasmid PUC57-BRC was constructed). The plasmid was identified by double enzyme digestion, and the copy number was approximately 3.3 × 10⁻⁶. 10 copies / μL.

[0042] Optimization of conditions for Brucella ddPCR method

[0043] (1) Primer and probe concentration optimization

[0044] The primer and probe concentrations of the ddPCR reaction system were optimized, with three parallel controls set up for each factor, as detailed below:

[0045] The working concentrations of both upstream and downstream primers were 1100 nmol / L, and the working concentration of the probe was 400 nmol / L.

[0046] The working concentrations of both upstream and downstream primers were 900 nmol / L, and the working concentration of the probe was 300 nmol / L.

[0047] The working concentrations of both upstream and downstream primers were 700 nmol / L, and the working concentration of the probe was 250 nmol / L.

[0048] The working concentrations of both upstream and downstream primers were 500 nmol / L, and the working concentration of the probe was 200 nmol / L.

[0049] The working concentrations of both upstream and downstream primers were 300 nmol / L, and the working concentration of the probe was 100 nmol / L.

[0050] The reaction system consisted of: 1.8 μL each of upstream and downstream primers; 0.6 μL of probe; 10 μL of ddPCR supermix; 1 μL of template; and water to a final volume of 20 μL. The reaction procedure is shown in Table 2.

[0051] Table 2 Brucella ddPCR reaction procedure

[0052]

[0053] Results Analysis: The results are as follows Figure 1 As shown, the reaction amplification efficiency is highest and the reaction system reaches its optimal state when the primer and probe concentrations are 900 / 300 nmol / L.

[0054] (2) Annealing temperature optimization

[0055] The annealing temperature of the ddPCR reaction system was optimized by setting five annealing temperatures: 50℃, 53℃, 55℃, 58℃ and 60℃, with three parallel controls for each factor.

[0056] The reaction system consisted of 1.8 μL each of upstream and downstream primers (900 nmol / L), 0.6 μL of probe (300 nmol / L), 10 μL of ddPCRsupermix, 1 μL of template, and water to a final volume of 20 μL. The reaction procedure is shown in Table 3.

[0057] Table 3 Optimized reaction procedures for Brucella annealing temperature

[0058]

[0059]

[0060] Results Analysis: The results are as follows Figure 2 As shown, the reaction amplification efficiency is highest and the reaction system reaches its optimal state when the annealing temperature is 60℃.

[0061] Example 2

[0062] (1) Sensitivity test and establishment of standard curve

[0063] The positive standard plasmid was diluted 30-fold, and then serially diluted 10-fold nine times (each 1.01 × 10⁻⁶). 8 1.01×10 7 1.01×10 6 1.01×10 5 1.01×10 4 1.01×10 3 1.01×10 2 1.01×10 1 1.01×10 0 (copies / μL), each gradient was measured 3 times. The mean of the last concentration point that could be detected in all samples was used as the sensitivity of the method;

[0064] The standard plasmid prepared in Example 1 was diluted to 1, 10, and 10... 2 10 3 10 4 The concentration was measured in copies / μL, and each dilution was tested three times to plot a standard curve.

[0065] Results Analysis: Sensitivity Results Figure 3 ) and standard curve ( Figure 4 The results show that this method has excellent sensitivity, with a detection limit of 1.01 copies / μL. The standard curve for this method is y = 1.0057x + 0.0028, R0. 2 =0.9994, indicating a good linear relationship.

[0066] (2) Specificity test

[0067] To verify the specificity of the ddPCR primer set and method constructed in this application, Escherichia coli O157:H7 (GDMCC 1.2702), Salmonella enterica (CMCC(B)50335), Salmonella typhi (CMCC(B)50071) were purchased from Guangdong Huankai Microbial Co., Ltd., Brucella antigen (Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.), Streptococcus suis (ATCC 700794, Beijing Bio-Bio Biotechnology Co., Ltd.), and Streptococcus agalactiae (ATCC 12386, Beijing Bio-Bio Biotechnology Co., Ltd.) were extracted and detected using the Brucella ddPCR method. The reaction system and reaction procedure were the same as those optimized in Example 1. The experimental results are as follows. Figure 5 As shown.

[0068] Results analysis: Except for Brucella antigen nucleic acid, no positive droplets were detected in the nucleic acid samples of other pathogens, indicating that the constructed Brucella ddPCR method has good specificity.

[0069] (3) Repeatability test

[0070] Brucella antigen nucleic acid was extracted, diluted 100-fold and 1000-fold, and detected using ddPCR. Each concentration was tested in triplicate. The standard deviation and coefficient of variation were calculated to evaluate the intra- and inter-group repeatability of the method (Table 4).

[0071] Table 4. Repeatability Tests of the ddPCR Method

[0072]

[0073]

[0074] Results analysis: As shown in Table 4, the coefficients of variation for the repeatability tests of the ddPCR method within and between groups were 2.8% to 5.1%, all of which were less than 10%, indicating that the established Brucella ddPCR method has good repeatability.

[0075] (4) Clinical sample testing

[0076] Thirty clinical samples collected from cattle and sheep slaughterhouses in Dongguan City were tested using the optimized Brucella ddPCR method and the real-time fluorescence PCR method (commercial kit), respectively, and the detection rates of the two methods were compared.

[0077] Results analysis: The results showed that the positive rate of Brucella by ddPCR was 16.67% (5 / 30), and the positive rate of real-time fluorescence PCR was also 16.67% (5 / 30), indicating that the detection rates of the two methods were consistent; the detection results of some clinical samples were as follows... Figure 6 and Figure 7 As shown.

[0078] Example 3

[0079] (1) Preparation of Brucella nucleic acid standards

[0080] The antigenic nucleic acid of Brucella agglutination antigen (Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.) was extracted from the red agglutination plate of Brucella, diluted 100-fold with nuclease-free water, aliquoted into 100 μL tubes, and stored at -80℃ for later use. Absolute quantification of nucleic acid was performed on three tubes using the Brucella ddPCR method established in this study, with each tube tested three times.

[0081] Results analysis: After 100-fold dilution of the antigen nucleic acid, the concentration of the target gene was 6240 copies / μL.

[0082] (2) Evaluation of the kit's lowest detection limit

[0083] Brucella nucleic acid standard (6240 copies / μL) was prepared using step (1), and 6 serial dilutions were performed at 4-fold gradients (1560 copies / μL, 390 copies / μL, 97.5 copies / μL, 24.4 copies / μL, 6.1 copies / μL, 1.5 copies / μL) (Table 5). The serially diluted Brucella standard was tested using three commercial Brucella fluorescent PCR detection kits. Each dilution was tested 5 times, and a detection rate of 95% was taken as the lowest detection limit of the kit.

[0084] Table 5. Limit of Detection Tests for Three Commercially Available Reagent Kits

[0085]

[0086] Results Analysis: The results of serially diluted Brucella standards tested using three commercially available kits are shown in Table 5. The limit of detection (LOD) of kit A was 24.4 copies / μL, while that of kits B and C was 97.5 copies / μL, indicating that kit A was superior to kits B and C.

[0087] (3) Evaluation of kit sensitivity and specificity

[0088] One hundred clinical negative samples were used, of which 20 samples were treated as known positive samples with 5-10 μL of Brucella antigen added. After nucleic acid extraction, the samples were amplified using three different kits. The sensitivity (Se) and specificity (Sp) of the kits were calculated based on the results. The calculation methods for sensitivity and specificity are shown in Table 6.

[0089] Table 6. Methods for calculating sensitivity and specificity

[0090]

[0091] Note: True positive (Tp), False positive (FP), True negative (TN), False negative (FN).

[0092] Sensitivity (Se) = TP / (TP+FN); Specificity (Sp) = TN / (TN+FP).

[0093] Table 7. Sensitivity and specificity results of three commercially available kits.

[0094]

[0095] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0096] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of Brucella virulence factor T4SS virB10 as a target sequence in the detection of Brucella in non-disease diagnosis and treatment.

2. A specific primer and probe set for ddPCR detection of Brucella, characterized in that, It includes a forward primer, a reverse primer, and a probe, with the specific sequences as follows: Forward primer: 5'-GGGGTTCAAATCAGATCAACCTC-3', SEQ ID NO.1; Reverse primer: 5'-GCGGAATGTTGATCGTATCCTTC-3', SEQ ID NO.2; Probe: 5'-CTCGTCGATTCACCTCCGCCGGTA-3', SEQ ID NO.3, wherein the 5' end of the probe is connected to a fluorescent reporter group and the 3' end is connected to a non-fluorescent quencher group.

3. The specific primer and probe set for ddPCR detection of Brucella according to claim 2, characterized in that, The fluorescent reporter group is FAM; the non-fluorescent quencher group is NFQ-MGB.

4. The use of the specific primer and probe set according to any one of claims 2-3 in the preparation of a kit for detecting Brucella.

5. A kit for detecting Brucella by ddPCR, characterized in that, Includes the specific primer and probe set as described in any one of claims 2 or 3.

6. The ddPCR detection kit for Brucella according to claim 5, characterized in that, The working concentrations of both the forward and reverse primers are 900 nmol / L; the working concentration of the probe is 300 nmol / L.

7. A method for detecting Brucella by ddPCR, characterized in that, Includes the following steps: (1) Extract DNA from the sample to be tested; (2) Using the DNA extracted in step (1) as a template, ddPCR amplification was performed using the primer and probe set described in any one of claims 2-3.

8. The method for detecting Brucella by ddPCR according to claim 7, characterized in that, The reaction system for ddPCR amplification is as follows: 1.8 μL each of 900 nmol / L upstream and downstream primers, 0.6 μL of 300 nmol / L probe, 10 μL of ddPCR supermix, 1 μL of template, and water to a final volume of 20 μL.

9. The method for detecting Brucella by ddPCR according to claim 7, characterized in that, The reaction procedure for ddPCR amplification is as follows: 95℃ for 10 min; 94℃ for 30 s, 60℃ for 1 min, 40 cycles; 98℃ for 10 min; store at 4℃.

10. The application of the primer and probe set according to any one of claims 2-3, the kit according to any one of claims 5-6, or the ddPCR method for detecting Brucella according to any one of claims 7-9 in the performance evaluation of commercial Brucella kits.

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