Animal skin disease detection kit

Abstract

The invention belongs to the technical field of animal epidemic disease detection, and provides a specific primer and a probe for fluorescent quantitative PCR (Polymerase Chain Reaction) detection of bacillus anthracis, and the specific primer and the probe comprise primers SEQ ID NO: 1-SEQ ID NO: 4 and probes SEQ ID NO: 5-SEQ ID NO: 6 on plasmids of bacillus anthracis pX01 and pX02. The invention also provides a detection kit containing the 2 * reaction buffer. The kit disclosed by the invention can be used for detecting the bacillus anthracis in the animal skin with high sensitivity and high specificity.

Description

Technical Field

[0001] The invention relates to the technical field of animal hide quarantine, belonging to the technical field of biotechnology. Background Art

[0002] my country is a major importer of hides and hairs. The types of imported hides and hairs include raw cowhide, wet-salt raw cowhide, salted raw cowhide, raw rabbit hide, rabbit hide, sheepskin and wet-salt sheepskin. The importing countries include the United States, Canada, Australia, New Zealand, France, Germany and Belgium.

[0003] Bacillus anthracis is recognized as one of the serious zoonoses and is a legal inspection item for import and export quarantine. At present, there are two types of detection methods for Bacillus anthracis. One is the traditional culture and its improved method, which relies on biochemical and morphological characteristics, has a long detection cycle and poor operability; the other is the polymerase chain reaction (PCR) related method, including ordinary PCR method and real-time fluorescence PCR method, which mainly targets multiple target genes that distinguish Bacillus anthracis from other bacteria of the genus Bacillus, selects specific sequences, and establishes PCR and real-time fluorescence PCR methods. Bacillus anthracis PCR-related detection methods have developed rapidly in recent years. Due to its rapid, sensitive and specific characteristics, it has become a reliable detection method.

[0004] Bacillus anthracis has the same morphological and biochemical characteristics as Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides and Bacillus pseudomycoides, and all belong to the Bacillus cereus group. The high similarity of bacteria leads to the possibility of horizontal gene transfer between species under appropriate temperature, humidity and pH conditions, and gene transfer leads to diagnostic difficulties. The OIE-recommended Bacillus anthracis PCR method uses the gene fragments on the virulence factor pXO1 and pXO2 plasmids as the detection object, and determines whether Bacillus anthracis is detected by detecting virulence factors. However, due to the existence of gene transfer, even the PCR method is difficult to distinguish Bacillus anthracis from other Bacillus cereus, and cross reactions often occur.

[0005] In recent years, in the monitoring of fur anthrax, the primers in the PCR diagnostic method recommended by OIE can cross-react with Bacillus cereus and Bacillus thuringiensis, which brings troubles to anthrax diagnosis. Therefore, there is an urgent need for a new anthrax Bacillus gene detection method in this field, which can eliminate the PCR cross-reaction between anthrax Bacillus and other cereus Bacillus and detect anthrax Bacillus quickly, stably and accurately. Summary of the invention

[0006] The purpose of the present invention is to solve the shortcomings of the prior art and to propose an animal skin disease detection kit and method.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides an animal skin disease detection kit, the kit comprising:

[0009] A first primer pair, wherein the first primer pair is a primer sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2;

[0010] A second primer pair, the second primer pair being the primer sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4;

[0011] A first probe, the first probe is SEQ ID NO: 5;

[0012] A second probe, the second probe is SEQ ID NO: 6;

[0013] and 2× reaction buffer.

[0014] The nucleotide sequences of the primers and probes are as follows:

[0015] SEQ ID NO:1 5'-TGGACCGATCCTCAAGATAA-3';

[0016] SEQ ID NO:2 5'-GTACCTAAAGAGTATAAGCA-3';

[0017] SEQ ID NO:3 5'-CGCAGCTATTAATATACCTGC-3';

[0018] SEQ ID NO:4 5'-AGGTTATGTGCCTTGAAGTT-3';

[0019] SEQ ID NO:5 5'-FAM-AGACCGTGACAATGATGGAATC-TAMRA-3';

[0020] SEQ ID NO:6 5'-CY5-CGTCTCATTCTACCTCACCTTAA-TAMRA-3'.

[0021] A further 2× reaction buffer consisted of the following components:

[0022] 10U / μl TaqDNA polymerase, 50mmol / L Tris-HCl, 20mmol / L KCl, 10mmol / L (NH4)2SO4, 0.1% Tween-20, 0.05% chitosan, 2.0mmol / L dNTPS, 2.0mmol / L sorbitol and 2mmol / L CoCl2 pH8.5.

[0023] In an embodiment of the present invention, the probe is modified with a fluorescent group, and the fluorescent group is selected from FAM, VIC, ROX or CY5. The 3' end of the probe is labeled with a quencher group, and the quencher group is MGB.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The detection kit developed by the present invention designs highly specific primers and probe sets and optimizes the reaction system, thereby improving the specificity and sensitivity of the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 is the fluorescence quantitative amplification curve of the experimental group in Example 4;

[0028] Figure 2 This is the fluorescence quantitative amplification curve of the control group in Example 4. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the examples. The equipment and reagents used in each embodiment and test example can be obtained from commercial sources unless otherwise specified. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0030] Based on the information contained in this application, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined process, properties or components, because these embodiments and other descriptions are only for illustrating specific aspects of the present invention. In fact, various changes that the technicians in this field or related fields can obviously make to the embodiments of the present invention are all covered within the scope of the appended claims.

[0031] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers used in this application to express the amount, percentage, and other numerical values ​​should be understood as modified by the word "approximately" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods. In the present invention, "about" means within 10% of a given value or range, preferably within 5%.

[0032] In the following embodiments of the present invention, when the temperature is not particularly limited, it is room temperature. Room temperature refers to the natural room temperature in four seasons without additional cooling or heating treatment, and is generally controlled at 10-30°C, preferably 15-25°C.

[0033] Unless otherwise specified, the reagents and materials used in the embodiments of the present invention can be purchased from the market.

[0034] The detailed implementation process and contents are as follows:

[0035] Example 1

[0036] The virulence of Bacillus anthracis mainly depends on two virulence encoding plasmids pX01 and pX02, among which edema factor (EF), lethal factor (LF), and protective antigen (PA) are encoded by virulence genes pag, lef, and cya located on pX01, respectively; the genes related to capsule formation are capB, capC, and capA on pX02. The conserved sequences of pX01 (NCBI Reference Sequence: NC_007322.2, SEQ ID NO: 6) and pX02 (GenBank: AB125962.1, SEQ ID NO: 7) genes of Bacillus anthracis were searched on GeneBank, and primers and probes were designed using Primer Explorer V4 software, which were synthesized by Shanghai BGI Genomics Co., Ltd. The optimal primer combination was screened out as follows:

[0037] SEQ ID NO:1 5'-TGGACCGATCCTCAAGATAA-3';

[0038] SEQ ID NO:2 5'-GTACCTAAAGAGTATAAGCA-3';

[0039] SEQ ID NO:3 5'-CGCAGCTATTAATATACCTGC-3';

[0040] SEQ ID NO:4 5'-AGGTTATGTGCCTTGAAGTT-3';

[0041] SEQ ID NO:5 5'-FAM-AGACCGTGACAATGATGGAATC-TAMRA-3';

[0042] SEQ ID NO:6 5'-CY5-CGTCTCATTCTACCTCACCTTAA-TAMRA-3'.

[0043] Example 2 Optimization of Fluorescence Quantitative PCR Reaction System and Specificity Detection

[0044] 1) Prepare different 2X reaction buffer solutions according to the following table:

[0045] Table 1 2× Reaction Buffer

[0046]

[0047] 2) The specific system was verified by artificially synthesizing plasmids based on the nucleic acid sequences of Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus weigeni, and Bacillus anthracis. The QPCR amplification system is shown in Table 2:

[0048] Table 2

[0049]

[0050]

[0051] Amplification conditions: 98℃5min [98℃15s, 60℃1min]*40

[0052] The test results are shown in Table 3. Reagents 3 to 6 all have good specificity.

[0053] Table 3

[0054]

[0055] Example 3 Sensitivity Detection

[0056] The concentrations were 5×10 5 , 5×10 4 , 5×10 3 , 5×10 2 , 5×10 1 , 5×10 0 , 3×10 0 , 1×10 0The synthetic plasmid of Bacillus anthracis with a concentration of 100 copies / μL was used as a template to perform fluorescence quantitative PCR for sensitivity detection. The reaction system and amplification conditions were as described in Example 2, and the results are shown in Table 4.

[0057] Table 4

[0058] Template concentration Reagent 3 Reagent 4 Reagent 5 Reagent 6 <![CDATA[5×10 5 ]]> Checkout Checkout Checkout Checkout <![CDATA[5×10 4 ]]> Checkout Checkout Checkout Checkout <![CDATA[5×10 3 ]]> Checkout Checkout Checkout Checkout <![CDATA[5×10 2 ]]> Checkout Checkout Checkout Checkout <![CDATA[5×10 1 ]]> Checkout Checkout Checkout Checkout <![CDATA[5×10 0 ]]> Checkout Checkout Checkout Checkout <![CDATA[3×10 0 ]]> Not detected Checkout Checkout Not detected <![CDATA[1×10 0 ]]> Not detected Not detected Checkout Not detected

[0059] Note: CT value ≤ 42 is the detection line

[0060] As can be seen from Table 4, the detection system established in the present application has a very high sensitivity and can well detect single-copy templates.

[0061] Example 4 Simulated fur sample detection

[0062] 3×10 5 CFU / mL anthrax vaccine strain spore suspension was diluted 10-fold and randomly added to 1g of cowhide sample with 200 μL each, shaken and mixed for 3 min, and placed at 4°C overnight to allow the spores to fully adsorb to the fur, which was contaminated fur. A total of 50 samples were prepared, of which 48 were added with anthrax, and the concentration of anthrax added was 3×10 5 CFU / mL~3×10 0 CFU / mL, 6 groups in total, 8 samples in each group, and 2 fur samples without bacteria added as negative controls.

[0063] Reagent 5 and reagent 6 in Table 1 of Example 2 were used as 2× buffer to prepare a reaction system, wherein reagent 5 was the experimental group and reagent 6 was the control group. The reaction system and QPCR amplification conditions were referred to Table 2, and fluorescence quantitative PCR was performed on 50 samples respectively, and whether anthrax Bacillus was present in the samples was determined based on the fluorescence quantitative PCR amplification curve.

[0064] The results are as follows Figure 1 and Figure 2 It shows that the experimental group ( Figure 1 ) detection rate was 95.8% (46 / 48), and the control group ( Figure 2 ) detection rate was 79.2% (38 / 48). The method established in this application has a high detection rate for anthrax Bacillus in fur samples.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An animal skin disease detection kit, characterized in that: The kit comprises: A first primer pair, wherein the first primer pair is a primer sequence shown in SEQ ID NO: 1 5'-TGGACCGATCCTCAAGATAA-3' and SEQ ID NO: 2 5'-GTACCTAAAGAGTATAAGCA-3'; A second primer pair, wherein the second primer pair is a primer sequence shown in SEQ ID NO: 3 5'-CGCAGCTATTAATATACCTGC-3' and SEQ ID NO: 4 5'-AGGTTATGGTGCCTTGAAGTT-3'; A first probe, wherein the first probe is SEQ ID NO: 5 5'-FAM-AGACCGTGACAATGATGGAATC-TA MRA-3'; The second probe is SEQ ID NO: 65'-CY5-CGTCTCATTCTACCTCACCTTAA-TAMRA-3'; and 2× reaction buffer.

2. The animal skin disease detection kit according to claim 1, characterized in that: The 2× reaction buffer consists of the following components: 10U / μl TaqDNA polymerase, 50mmol / L Tris-HCl, 20mmol / L KCl, 10mmol / L (NH4)2SO4, 0.1% Tween-20, 0.05% chitosan, 2.0mmol / L sorbitol and 2mmol / L CoCl2, 2.0mmol / L dNTPSPH8.

5.

3. The animal skin disease detection kit according to claim 1, characterized in that: The probe is modified with a fluorescent group, and the fluorescent group is selected from FAM, VIC, ROX or CY5.

4. The animal skin disease detection kit according to claim 1, characterized in that: The 3' end of the probe is labeled with a quencher group, and the quencher group is MGB.

5. Use of the kit according to claim 1-4, characterized in that: Use of a kit for preparing a kit for detecting anthrax bacillus.

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