Digital polymerase chain reaction (PCR) method of microdroplet type based on Bacillus anthracis

Description

2 and the capAdespXO2 plasmid associated with capsule synthesis were used as target genes for detection, and the detection sensitivity of the capA gene and the PA gene reached 5 and 50 copies / µl respectively, thereby increasing the sensitivity of Bacillus anthracis detection. While the aforementioned methods can detect the pathogenicity factor pXO2,5 existing techniques for detecting the pathogenicity factor pXO2 still have the following disadvantages: The existing methods are unable to detect trace amounts of Bacillus anthracis at low concentrations in the environment and in diseased material. Currently, there is no microdroplet-type digital polymerase chain reaction (PCR) detection method for Bacillus anthracis, and10 the detection sensitivity needs to be further improved. Moreover, detection via PCR methods only allows for qualitative analysis; quantitative analysis is not possible.Therefore, the development of a method for detecting trace amounts of pathogenicity factor spXO2 is of crucial importance for research on the pathogenicity factor of Bacillus anthracis.15 CONTENT OF THE PRESENT INVENTION The purpose of this part is to describe some aspects of the embodiments of the invention and to briefly present some preferred embodiments. In this part, as well as in the summary of the description and the title of the invention, 20 simplifications or omissions may be made to avoid any ambiguity regarding the purpose of this part, the summary of the description and the title of the invention, provided that these simplifications or omissions may not be used to limit the scope of the present invention. To solve the problem of the state of the art, namely that no trace amounts of Ba-25 cillus anthracis can be detected in low concentrations in the environment and in diseased material, the present invention uses the following technical solutions.A digital polymerase chain reaction (PCR) method of the microdroplet type based on Bacillus anthracis comprises the following steps: 30 Extraction of a DNA template: Extracting a DNA template from Bacillus anthracis, including a pXO2 DNA template; PCR amplification: Adding the pXO2 template to the PCR reaction system, performing a microdroplet treatment, and then performing PCR amplification in the order of pre-denaturation, denaturation, and attachment; and 35 BE2025 / 5531 3 Qualitative and quantitative analysis: Performing a qualitative and quantitative analysis of the system after PCR amplification. Preferably, the PCR reaction system in the above-mentioned microdroplet-type digital PCR method comprises the following: a 2x Supermix reaction solution; a pXO2-F primer; a pXO2-R primer; a 5 pXO2 probe; and the pXO2 DNA template and a DEPC water. Preferably, the above-mentioned microdroplet-type digital PCR method includes a gene sequence of the pXO2-F primer as in SEQIDNO.Figure 1 shows a gene sequence of the pXO2-R primer as shown in SEQIDNO.2; and Figure 10 shows a gene sequence of the pXO2 probe as shown in SEQIDNO.3. Preferably, the PCR reaction system in the above-mentioned microdroplet-type digital PCR procedure comprises the following: 2 x Supermix reaction solution 15 µl; pXO2-F primer 1.2 µl; 15 pXO2-R primers 1.2 µl; pXO2 probe 0.9 µl; pXO2 DNA template 2 µl; and DEPC water 9.7 µl. Preferably, the steps of the microdroplet treatment in the above-mentioned digital PCR method of the microdroplet type are as follows: Adding a reaction system into the eight wells of the first row of a DG8 well plate, and adding a microdroplet generating oil into the second row of the DG8 well plate, sealing the plate by means of a microdroplet generating rubber seal, and inserting the well plate with the reaction system and the microdroplet generating oil into a droplet generator for microdroplet treatment to form water-in-oil microdroplets.Preferably, the steps of the qualitative and quantitative analysis in the above-mentioned digital PCR method of the microdroplet type are as follows: using a digital PCR microdroplet analyzer to perform VIC-30 channel detection on the microdroplet in the well plate and to record a proportion of positive microdroplets; and detecting a pXO2 gene sequence by VIC. The present invention also provides, on the other hand, a method for the sensitivity test of the above-mentioned digital PCR method of the microdroplet type, comprising the following steps: BE2025 / 5531 4 Construction of the plasmid: including constructing an pXO2 plasmid; Plasmid dilution: Dilute the pXO2 plasmid in equal proportions; and PCR detection: Perform a digital PCR detection of the diluted pXO2 plasmid. Preferably, the steps of constructing the pXO2 plasmid in the above-mentioned 5 susceptibility testing procedure are as follows: Using a SEQIDNO.4. Using the gene sequence plasmid shown in SEQIDNO.5 as a template, inserting a VirB8 gene sequence shown in SEQIDNO.5 into the PUC plasmid to construct the pXO2 plasmid. Preferably, the concentration of the spXO2 plasmid after dilution in the above-mentioned sensitivity test procedure is as follows: 1.0 x 10⁵ copies / μl, 1.0 x 10⁴ copies / μl, 1.0 x 10⁳ copies / μl, 1.0 x 10⁲ copies / μl, 1.0 x 10⁹ copies / μl, 0.5 x 10⁹ copies / μl, 0.25 x 10⁹ copies / μl, 0.125 x 10⁹ copies / μl, 0.1 x 10⁹ copies / μl.In comparison to the prior art, the present invention exhibits the following advantageous effects: 15 The present invention provides a digital microdroplet-type PCR method based on Bacillus anthracis, in particular for the detection of pXO2 in Bacillus anthracis, comprising the following steps: First, a PCR amplification of pXO2 is carried out, and the amplified system is fed into a digital PCR analyzer for single-channel detection in order to detect pXO2; furthermore, the detection method according to the invention can detect the proportion of positive microdroplets of pXO2 and thus enables, on the one hand, a quantitative analysis of pXO2 and, on the other hand, a quantitative analysis of pXO2. The inventive digital PCR method of the microdroplet type is subjected to a sensitivity analysis during application in order to test the sensitivity of the method.Specifically, a standard plasmid is produced and a gradient dilution of the plasmid with a standard concentration is performed; by PCR testing and analysis on standard plasmids of different concentrations, the detection limit of the method can be determined, thereby analyzing the sensitivity of the method and applying the method to technologies with different testing requirements. This ensures the sensitivity of the method on the one hand and expands the application range of the method on the other. Furthermore, the PCR method of the present invention also offers a specific analytical method. Specifically, the method is used with other bacterial species such as Escherichia coli as controls for the analysis and detection of Bacillus anthracis 35 and other control bacteria. The results show that the method can effectively detect Bacillus anthracis.To further ensure the accuracy of the specific method, Bacillus anthracis is mixed with other bacterial species according to the invention and subsequently detected. If the individual control bacterial species is not expressed, but the mixed bacterial species is already expressed, it is ensured that the primers and probes in the specific experiment can effectively express the target bacterial species, which further ensures the accuracy of the detection method and clarifies the effectiveness of the detection method. DESCRIPTION OF THE DRAWING Fig. 1 is a VIC scatter plot from the sensitivity test of the microdroplet-type digital PCR method of the present invention; and Fig. 2 is a VIC scatter plot from the specificity test of the microdroplet-type digital PCR method of the present invention.DETAILED DESCRIPTION 15 In order to make the above-mentioned objectives, features and advantages of the present invention clearer and more understandable, the specific embodiments of the present invention are described in detail below with reference to the attached drawings. The following description serves to improve understanding of the present invention and contains many specific details. However, the present invention can also be implemented in ways other than those described here. People skilled in the art can make similar extensions without infringing the meaning of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. Secondly, the term "embodiment" or "design" here refers to a specific feature, structure, or property that may be included in at least one embodiment of the present invention.The expression “in one embodiment”, which appears in various places in this description, does not necessarily refer to the same embodiment, nor to a single or selected embodiment that excludes other embodiments. The present invention provides the following embodiments. The present invention provides a method for detecting the pathogenicity factor pXO2. Specifically, the digital microdroplet PCR method is used to detect the pathogenicity factor pXO2, thereby overcoming the deficiency of the state of the art 35 that it is not possible to detect trace amounts of Bacillus anthracis at low concentrations BE2025 / 5531 6. The method detects the pathogenicity factor pXO2 using the digital microdroplet PCR method and comprises in particular the following steps: First, the probe for pXO2 is designed and synthesized, and the specific primer-probe sequences of pXO2 are as follows: 5 pXO2-F:5'-GAGGCTCTTGGGATTGATGAG-3'(SEQIDNO.1) pXO2 probe:(VIC)5'-TCCGTGGTATGTTGAATGCTCATCCG-3'(BHQ1;SEQIDNO.3) pXO2-R:5'-CGAAGAACGCAGGCTTAGAT-3'(SEQIDNO.2); Secondly, various samples are collected as follows: Taking from the Bacillus anthracis-infected animal tissue or diseased material 10 and placing it in a 2-ml centrifuge tube, crushing it and adding an appropriate amount of sterile PBS, adding steel balls and vibrating for 2 minutes at a frequency of 20–30 Hz, then removing the steel balls, subsequently repeatedly freezing and thawing the diseased material three times in a refrigerator at -80°C, then centrifuging for 5 minutes in a centrifuge running at a speed of 10,000 rpm, storing samples after completion of the above procedures for later use in a refrigerator at -80°C.Taking a swab from the environment of Bacillus anthracis and placing it in a 2 ml centrifuge tube, and adding an appropriate amount of sterile PBS and mixing well, followed by repeated freezing and thawing of the diseased material 20 times in a refrigerator at -80°C, and storing samples after completion of the above procedures for later use in a refrigerator at -80°C. In addition, a digital PCR procedure is performed on the DNA template, comprising the following steps: The first step consists of extracting DNA template from the collected samples - 25 the DNA of Bacillus anthracis is extracted from the above-mentioned samples using a kit procedure and labeled as DNA template. The second step consists of setting up a digital PCR reaction system. Specifically, 15 µl of 2x Supermix reaction solution, 1.2 µl of the pXO2-F primer shown in SEQIDNO.1, 1.2 µl of the pXO2-R primer shown in SEQIDNO.2, and 0.9 µl of SEQIDNO.30 µl of the pXO2 sample probe, 2 µl of the pXO2 DNA template and 9.7 µl of the DEPC water were added to a sterilized PCR reaction tube to set up a digital PCR reaction system. The total volume of the reaction system is 30 µl. The third step is the microdroplet treatment. The above-mentioned reaction system is added to the eight wells of the first row of a DG8 well plate, and 35 180 µl of microdroplet generating oil are added to the second row of the DG8 well plate BE2025 / 5531 7. The plate is then sealed with a microdroplet generating rubber gasket. The well plate with the above-mentioned reaction system and the microdroplet generating oil is placed in a microdroplet generator, and by running a computer program for 300 seconds, microdroplets are generated that form oil-in-water microdroplets. In this embodiment, the oil is a continuous phase and the water is a dispersed phase in the oil-in-water microdroplets.In the PCR reaction system of this embodiment, the continuous phase is located on the surface of the dispersed phase and together they form microdroplets, so that the dispersed phase does not spread into the continuous phase and thus the effect of the subsequent PCR amplification is ensured.10 The fourth step is the PCR amplification, which is carried out according to the reaction conditions shown in Table 1. Table 1: PCR Reaction Conditions Step Temperature Time Number of Cycles 1 Pre-Denaturation 95°C 10 min 1 2 Denaturation 94°C 30 sec 40 Attachment, Spread 60°C 60 sec 3 Cooling of the Instrument 12°C 5 min 1 15 It is noteworthy that the pre-denaturation of the above-mentioned PCR amplification is carried out under conditions of 95°C and a time of 10 minutes. In this configuration, preferably five cycles are carried out under conditions of 95°C and a time of 10 minutes, i.e.The number of matrices in the PCR amplification system is 25 times that of the original matrices, thereby achieving more effective unwinding of the double-stranded matrices. In this configuration, the degree of unwinding of pXO2 can be increased by five pre-denaturing steps, which supports PCR amplification during the denaturation process, improves the accuracy of the PCR amplification results, and ensures the accuracy of the PCR procedure in this configuration. The fifth step is the qualitative and quantitative analysis of the above-mentioned system after the amplification reaction. Specifically, the amplified DG8 well plate is inserted into the digital PCR microdroplet analyzer, and the microdroplet data are The digital PCR microdroplet analyzer automatically aspirates the microdroplets from the well plate and performs VIC-30 BE2025 / 5531 8-channel detection to verify the pXO2 gene sequence. As shown in Fig. 1.

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