CN117778600A - Yersinia pestis triple-droplet digital PCR detection primer probe combination and detection method - Google Patents
Yersinia pestis triple-droplet digital PCR detection primer probe combination and detection method Download PDFInfo
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Abstract
The invention discloses a yersinia pestis triple-droplet digital PCR detection primer probe combination and a detection method. The method of the invention uses caf1 and plat on Yersinia pestis plasmids and ypo2088 genes on chromosomes as target genes, and the used composition consists of a multiplex PCR primer pair composition (A, B and C) and a probe composition (P1, P2 and P3); primer pair A and probe P1 can specifically bind ypo2088; primer pair B and probe P2 can specifically bind caf1; primer pair C and probe P3 can specifically bind to pla. The method can be based on a digital PCR instrument with a double-color fluorescent channel, can detect three target genes simultaneously in a single-tube reaction system, has detection sensitivities of 32 fg/mu L, 10 fg/mu L and 1 fg/mu L respectively, and can be applied to high-sensitivity detection of trace Yersinia pestis in complex samples such as soil, animal organ tissues and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a yersinia pestis triple-droplet digital PCR detection primer probe combination and a detection method.
Background
Plague is a virulent natural epidemic infectious disease caused by Yersinia pestis (hereinafter referred to as "plague"). The pestis has strong infectivity and high mortality, and early detection and diagnosis are very important for the treatment of pestis cases and the control of epidemic situations. Molecular biological detection methods based on polymerase chain reaction (polymerase chain reaction, PCR) have been widely used for rapid, high-sensitivity detection of plague bacteria. The technology uses caf1 and pla genes on plague bacillus plasmids as target genes for detection. Wherein, the caf1 gene is located on the pMT1 plasmid, and the pla gene is located on the pPCP1 plasmid. The plasmid is an unstable genetic material, and the isolation of plague bacteria with deletion of pPCP1 plasmid and plague bacteria with deletion of pMT1 plasmid is reported at present; therefore, there may be a pestilence detection miss with the plasmid gene as a target. In view of this, the world health organization (World Health Organization, WHO) revised the definitive definition of pestis cases in 2021 and recommended 4 target genes as pestis PCR detection targets, including caf1 and pla genes on pestis plasmids, and ypo2088 and ypo2486 genes on chromosomes; and, at least two target genes are required to be positive when the plague PCR detection is carried out, so that the condition of plague detection is met.
In general, animal carcasses with unknown causes of death occurring in epidemic fields and contaminated soil around the animal carcasses are the most common samples for detecting and isolating plague bacteria. Both sample types typically contain a large amount of PCR reaction inhibitor, which has a certain impact on the detection performance of the PCR detection method. Researchers establish an isothermal amplification PCR method for detecting plague bacteria, and evaluate various simulated sample types; as a result, it was found that the sensitivity of detection of plague bacteria in the soil sample and the mouse organ tissue sample was reduced by 10-fold and 1000-fold, respectively, compared with that in the blood sample (related documents: you Y, zhang P, wu G, tan Y, zhao Y, cao S et al Highly Specific and Sensitive Detection of Yersinia pestis by Portable Cas a-UPTLFA platform. Front in microbiology.2021;12.doi: 10.3389/fmicb.2021.700016.). Therefore, how to realize the high-sensitivity detection of trace pestis in complex samples such as soil and animal organ tissues has important practical significance for improving the detection accuracy of pestis.
Microdroplet Digital PCR (ddPCR) is a current new generation of nucleic acid detection technology, has higher detection sensitivity and better sample tolerance compared with the traditional PCR technology, and can realize absolute quantitative analysis independent of standard substances and standard curves, so that the method is particularly suitable for high-sensitivity detection of trace pestis in complex samples. There are studies reported to detect plague bacteria based on digital PCR, however, the method uses only one specific fragment on plague bacteria chromosome as target gene, and does not involve detection of other target genes.
Disclosure of Invention
The technical problem to be solved by the invention is how to prepare a product for detecting Yersinia pestis with high sensitivity and high sample tolerance and/or how to prepare a product for detecting Yersinia pestis with trace amount in a complex sample and/or how to detect Yersinia pestis with high sensitivity and high sample tolerance and/or how to detect Yersinia pestis with trace amount in a complex sample and/or how to detect Yersinia pestis trace amount in soil or animal organ tissue samples.
In order to solve the technical problems, the invention firstly provides a composition based on microdroplet digital PCR detection or auxiliary detection of Yersinia pestis (Yersinia pestis). The composition may consist of a multiplex PCR primer pair composition and a probe composition. The multiplex PCR primer pair may consist of primer pair a, primer pair B, and primer pair C. The probe composition may consist of three probes, P1, P2 and P3.
A fluorescent group may be labeled on the 5' nucleotide of the nucleotide sequence of each of the three probes. The 3' -terminal nucleotide of the nucleotide sequence of each probe may be labeled with a quenching group. The primer pair A can be specifically bound to a target gene ypo2088 on a yersinia pestis genome chromosome, the primer pair B can be specifically bound to a yersinia pestis genome plasmid target gene pla, and the primer pair C can be specifically bound to a yersinia pestis genome plasmid target gene caf1. The 5' nucleotides of P1 and P2 may be labeled with the same fluorescent group, and P3 and P1 and P2 may be labeled with different fluorescent groups. In the above composition, the fluorescent group may be selected from, but not limited to, common fluorescent groups such as FAM (5/6-carboxyfluorescein), VIC, TET (tetra-chloro-6-carboxyfluorescein), JOE (2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein), HEX (hexachloro-6-methylfluorescein), cy3, TAMRA (6-carboxytetramethyl rhodamine), ROX (carboxy-X-rhodamine), texas Red, LC Red640, cy5 (cyanine fuel), LC Red705, FITC (fluorescein isothiocyanate), and the like.
The quenching group can be selected from at least one of TAMRA, BHQ1, BHQ2, BHQ3, MGB and Dabcy 1.
In the above composition, the molar ratio of P1 to P2 may be 2 or more: 1.
in the above composition, the P1 can specifically bind to the target gene ypo2088 on the chromosome of Yersinia pestis genome, and the final concentrations of P1, P2 and P3 in the multiplex PCR system can be 500nM, 250nM and 250nM-1500nM, respectively. The final concentration of P3 in the multiplex PCR system may be specifically 250nM.
In the composition, the primer pair A can be a primer pair consisting of single-stranded DNA shown in a sequence 1 and single-stranded DNA shown in a sequence 2 in a sequence table; the primer pair B can be a primer pair consisting of single-stranded DNA shown in a sequence 3 and single-stranded DNA shown in a sequence 4 in a sequence table; the primer pair C can be a primer pair consisting of single-stranded DNA shown in a sequence 5 and single-stranded DNA shown in a sequence 6 in a sequence table. The nucleotide sequence of the probe P1 can be a sequence 3 in a sequence table; the nucleotide sequence of the probe P2 can be a sequence 6 in a sequence table; the nucleotide sequence of the probe P3 can be a sequence 9 in a sequence table.
In order to solve the above technical problems, the present invention also provides a reagent and/or kit for identifying or assisting in identifying Yersinia pestis (Yersinia pestis), which may contain the composition as described above.
In order to solve the above technical problems, the present invention also provides a system for identifying or aiding in the identification of Yersinia pestis (Yersinia pestis), said system comprising the above-described reagents and/or kits.
In the above system, the system may further comprise a digital PCR system.
In order to solve the above technical problems, the present invention also provides a method for detecting or assisting in detecting Yersinia pestis (Yersinia pestis), the method comprising performing a digital micro-droplet PCR analysis on a sample to be tested with the composition described above, or the reagent or kit described above, or the system described above, and determining or assisting in determining whether the sample to be tested is Yersinia pestis (nucleic acid) or contains Yersinia pestis (nucleic acid) or is infected with Yersinia pestis based on the digital micro-droplet PCR detection result.
In the above method, the molar ratio of P1 and P2 in the microdroplet digital PCR system may be 2 or more: 1. the concentrations of P1, P2 and P3 in the microdroplet digital PCR system may be 500nM, 250nM and 250nM-1500nM, respectively. The concentration of said P3 in said microdroplet digital PCR system may in particular be 250nM.
In order to solve the technical problems, the invention also provides application of the composition in preparation of yersinia pestis detection or auxiliary detection products. The product can be a product for detecting complex samples such as soil or animal organs.
The target gene ypo2088 may have a Genbank number of 1174927, the target gene pla may have a Genbank number of 57977666, and the target gene caf1 may have a Genbank number of 57977636.
The above-described applications or methods are non-disease diagnostic applications or methods. The above applications or methods may not be directed to obtaining disease diagnosis results or health status of a living human or animal body. The sample to be tested may be a sample from a non-living human or animal body, such as an environmental sample (e.g. water), a food (e.g. frozen food or fresh food).
The invention establishes a high-sensitivity pestis detection method based on microdroplet digital PCR by taking caf1 and pla on a pestis plasmid and ypo2088 gene sequences on a chromosome as targets. The method can be based on a digital PCR instrument with a double-color fluorescent channel, can detect three specific target genes of pestis simultaneously in a single-tube reaction system, and can realize high-sensitivity detection of trace pestis in complex samples.
The plague bacteria digital PCR detection method established by the invention has the following advantages compared with other detection methods:
(1) And (5) multi-gene combined detection. The currently reported plague digital PCR detection method only aims at one target gene on chromosome, and the plague digital PCR detection target genes established by the invention cover three target genes of plague caf1, plat and ypo 2088. In addition, the method can simultaneously realize the joint detection of the three target genes in a single-tube reaction system by using a common double-color fluorescent microdroplet digital PCR instrument, so that the operation efficiency can be improved, the reagent loss can be reduced, and the requirement of the method on the digital PCR instrument can be reduced.
(2) The detection sensitivity is higher. Compared with the traditional real-time fluorescent PCR detection method, the digital PCR detection method for the pestis established by the invention has the advantage that the detection sensitivity can be improved by 10-100 times, so that the digital PCR detection method for the pestis is more suitable for high-sensitivity detection of trace pestis in complex samples.
Drawings
Figure 1 shows the results of single target droplet digital PCR detection of plague. (A) The detection result of the single-target digital PCR one-dimensional scatter diagram is that the abscissa is the target gene, and the ordinate is the fluorescence intensity (Amplitude) of the liquid drop; (B) The average fluorescence intensity of the positive liquid drops under the conditions of different probe concentrations is shown in the abscissa, the probe concentration is shown in the ordinate, and the average fluorescence intensity of the positive liquid drops is shown in the ordinate (Mean amplitude of positives).
FIG. 2 shows the detection results of a pestivirus multi-target droplet digital PCR two-dimensional scattergram. The abscissa is the second fluorescence Channel (Channel 2) detection signal intensity, and the ordinate is the first fluorescence Channel (Channel 1) detection signal intensity.
FIG. 3 shows the results of the multi-target digital PCR sensitivity evaluation of the present invention. (A) The detection sensitivity evaluation result of the multi-target digital PCR on the ypo2088 target genes is obtained; (B) The detection sensitivity evaluation result of the caf1 target gene for multi-target digital PCR; (C) And (3) evaluating the detection sensitivity of the multi-target digital PCR to the pla target genes.
FIG. 4 is a comparison of the detection performance of the multi-target digital PCR of the present invention and real-time fluorescent PCR on liver tissue spiked simulated samples. (A) The detection sensitivity result of the multi-target digital PCR of the invention on the target gene ypo2088 in the sample is obtained; (B) Detecting a sensitivity result of the microdroplet digital PCR of the invention on a target gene caf1 in a sample; (C) The detection sensitivity result of the multi-target digital PCR on the target gene pla in the sample is obtained; (D) The detection sensitivity result of the target gene ypo2088 in the sample is obtained by single-target real-time fluorescence PCR; (E) The detection sensitivity result of the single-target real-time fluorescence PCR on the target gene caf1 in the sample is obtained; (F) The detection sensitivity result of the single-target real-time fluorescence PCR on the target gene pla in the sample is obtained.
FIG. 5 is a comparison of the detection performance of the multi-target digital PCR of the present invention and real-time fluorescent PCR on soil spiked simulated samples. (A) The detection sensitivity result of the multi-target digital PCR of the invention on the target gene ypo2088 in the sample is obtained; (B) Detecting a sensitivity result of the microdroplet digital PCR of the invention on a target gene caf1 in a sample; (C) The detection sensitivity result of the multi-target digital PCR on the target gene pla in the sample is obtained; (D) The detection sensitivity result of the target gene ypo2088 in the sample is obtained by single-target real-time fluorescence PCR; (E) The detection sensitivity result of the single-target real-time fluorescence PCR on the target gene caf1 in the sample is obtained; (F) The detection sensitivity result of the single-target real-time fluorescence PCR on the target gene pla in the sample is obtained.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Yersinia pestis (Yersinia pestis), yersinia pseudotuberculosis (Yersinia pseudotuberculosis), yersinia enterocolitica (Yersinia enterocolitica), yersinia ruckeri (Yersinia ruckeri), yersinia ruckeri (Yersinia kristensenii), yersinia freundii (Yersinia frederiksenii), yersinia moensis (Yersinia mollaretii) and Luo Shiye Yersinia rohdei) are the laboratory preservations in the examples of the present invention, and the relevant literature: du Y, yan Z, song K, jin J, xiao L, sun Z et al development and evaluation of a multiplex droplet digital polymerase chain reaction method for simultaneous detection of five biothreat pathens. Doi:10.3389/fmib.2022.970973.
Example 1, construction of Yersinia pestis Single target microdroplet digital PCR detection System
1. Primer design and probe synthesis
According to the literature search, the amplification primer pair and the detection probe of the yersinia pestis chromosome target gene ypo2088 and the plasmid target gene pla, and the amplification primer and the detection probe of the plasmid target gene caf1 are obtained.
TABLE 1 primer and probe sequences for detection of Yersinia pestis
The primers and probes used were all synthesized by Shanghai Biotechnology, inc., wherein the primers were PAGE purified and the probes were HPLC purified. The fluorescent probes used are double-labeled probes: labeling a fluorescent group at the 5' end; the 3' end marks the quenching group. In the embodiment, HEX fluorescent labels are adopted at the 5 'ends of the probes P1, P2 and P3, and BHQ-1 quenching groups are labeled at the 3' ends (namely, a 1 st nucleotide modified HEX fluorescent group, a 22 nd nucleotide modified BHQ-1 quenching group, a 1 st nucleotide modified HEX fluorescent group, a 24 th nucleotide modified BHQ-1 quenching group, a 1 st nucleotide modified HEX fluorescent group and a 24 th nucleotide modified BHQ-1 quenching group of a sequence 3 in a sequence table), and the 1 st nucleotide modified BHQ-1 quenching groups of a sequence 9 are adopted.
2. Bacterial genome template preparation
After inactivating the overnight culture of Yersinia pestis, DNA was extracted separately using QIAamp DNA Mini Kit (Qiagen 51304) and finally eluted with 100. Mu.L TE buffer.
Preparation of ddPCR reaction System
In the embodiment, the invention respectively establishes a single-target gene microdroplet digital PCR detection system aiming at three target genes of yersinia pestis ypo2088, caf1 and pla. The total volume of each ddPCR reaction system is 20 mu L, wherein the DNA of the sample to be detected is 2 mu L; the final concentrations of the detection probes P1, P2 and P3 were 100 to 1500nM (100 nM, 250nM, 500nM, 1200nM, 1500 nM), and the insufficient volumes were filled with ultrapure water, and the specific reaction system and component concentrations were as shown in Table 2:
TABLE 2 final concentration of major Components of ddPCR reaction System
Note that: in Table 2, the primers are F1 and R1, and the probe is P1; or the primer is F2 and R2, and the probe is P2; or the primer is F3 and R3, and the probe is P3.
1.4 droplet Generation
After the ddPCR amplification system prepared in the step 1.3 is uniformly mixed, adding the ddPCR amplification system into a sample hole of a Droplet generator (Bio-Rad QX200 Droplet), adding 70 mu L of Droplet generation oil into an oil hole, covering a gasket, and placing the mixture into the Droplet generator to prepare droplets;
1.5PCR amplification
Transferring 40 mu L of microdroplets to a 96-well PCR plate, and performing multiple ddPCR amplification on a Bio-Rad T100 PCR instrument after membrane sealing, wherein the PCR reaction conditions are as follows: pre-denaturation at 95℃for 10min, 30sec at 95℃and 1min at 60℃for 40 cycles.
1.6 interpretation of results
After the amplification is completed, transferring the PCR reaction plate to a Bio-Rad QX200Droplet Reader, and performing Droplet counting and fluorescent signal detection according to the operation instructions of an instrument and software; meanwhile, the droplets are classified and counted by using Bio-Rad QuantaSoft Analysis Pro version 1.0.596 software; and finally, carrying out positioning division on the generated droplet clusters by using a two-dimensional scatter diagram.
As a result, as shown in fig. 1 (a), the positive droplet clusters and the negative droplets of ypo2088 (left panel of fig. 1 (a)) and caf1 gene (middle panel of fig. 1 (a)) can be well distinguished, and the positive droplet clusters are dense and non-dispersed; the positive droplet clusters were more dispersed in the amplification result of the pla gene, i.e., the "raindrop" phenomenon was present (right panel of FIG. 1 (A)).
As shown in the results (B) in FIG. 1, as the concentration of the detection probe in the system increases, the fluorescence intensity of the positive droplets of ypo2088 and caf1 genes also gradually increases; while the positive droplet fluorescence intensity of the pla gene did not show a better linear increase.
In view of the above two points, in the multi-target detection system, probe P3 of the pla gene was fluorescently labeled with HEX (or FAM), while probes of ypo2088 and caf1 genes were fluorescently labeled with FAM (or HEX).
Under the condition of the same probe concentration, the fluorescence intensity of positive microdroplets of the ypo2088 gene is obviously higher than that of the caf1 gene, and when the final concentration of a ypo2088 gene detection probe (P1) is 500nM and the final concentration of a caf1 gene detection probe (P2) is 250nM, positive microdroplet clusters of two target genes can be well separated, and the dosage is the most economical. When the final concentration of the pla gene detection probe (P3) is 250nM, the positive droplet cluster and the negative droplet cluster can be separated well. Thus, in the multi-target detection system, the final concentration of the probe (P1) of the ypo2088 gene was set to 500nM, the final concentration of the caf1 gene detection probe (P2) was set to 250nM, and the final concentration of the pla gene detection probe (P3) was set to 250nM.
Example 2, establishment of method for rapid detection of Yersinia pestis based on Multi-target microdroplet digital PCR the method for rapid detection of Yersinia pestis based on Multi-target microdroplet digital PCR of this example is different from example 1 in that HEX fluorescent label modification is adopted at the 5 'end of the detection probe P1 and P2 sequences in this example (i.e. HEX fluorescent group modified at nucleotide 1, BHQ-1 quenching group modified at nucleotide 22, HEX fluorescent group modified at nucleotide 1, BHQ-1 quenching group modified at nucleotide 24 of sequence 6) and FAM fluorescent label modification is adopted at the 5' end of the detection probe P3 sequence (i.e. FAM fluorescent group modified at nucleotide 1, BHQ-1 quenching group modified at nucleotide 24 of sequence 9 in the sequence table); the final concentration of probe P1 in the reaction system was 500nM, the final concentration of probe P2 was 250nM, and the final concentration of probe P3 was 250nM; the specific reaction system and the component concentrations are shown in table 3:
TABLE 3 final concentrations of major Components of Yersinia pestis Multi-target microdroplet digital PCR reaction System
The primer consists of 6 primers of F1, R1, F2, R2, F3 and R3, and the concentration of each primer is 900nM; the remaining steps remain the same as in example 1.
The two-dimensional scatter diagram results are shown in fig. 2, and the detection results of three target genes can be well distinguished, and the pla gene has raindrop phenomenon, but does not interfere with the amplification results of ypo2088 gene and caf1 gene of the other channel; the microdroplet clusters of ypo2088 gene and caf1 gene are in the same fluorescent channel, but can be well distinguished according to the fluorescent intensity of the microdroplet clusters due to the probe concentration difference.
Example 3 sensitivity evaluation test
After inactivating the overnight culture of Yersinia pestis, DNA was extracted with QIAamp DNA Mini Kit (Qiagen 51304) and finally eluted with 100. Mu.L TE buffer. The DNA solution concentration obtained by measurement of a Thermo Nanodrop concentration tester is subjected to 10-time gradient dilution by using a TE buffer to obtain DNA solutions with different concentrations of 1 fg/. Mu.L to 0.1 ng/. Mu.L. Sensitivity evaluation was performed using genomic DNA after extraction and purification of plague as a template, and sensitivity evaluation experiments were performed using the method for rapid detection of yersinia pestis based on multi-target microdroplet digital PCR in example 2.
As shown in FIG. 3, the detection sensitivity of Yersinia pestis multi-target microdroplet digital PCR of the present invention to the target genes ypo2088, caf1, and pla was 32 fg/. Mu.L (FIG. 3 (A)), 10 fg/. Mu.L (FIG. 3 (B)), and 1 fg/. Mu.L (FIG. 3 (C)), respectively, using TE buffer as a negative control, and the detection result was a blank detection Limit (LOB).
Example 4 specificity evaluation test
Yersinia pestis was detected by the method based on multi-target microdroplet digital PCR set up in example 2, wherein the difference from example 1 is that 7 species of Yersinia pestis (including Yersinia pseudotuberculosis, yersinia enterocolitica, yersinia ruckeri, yersinia keri, yersinia freudenri, yersinia mohni and Luo Shiye Yersinia pestis) were selected for the specificity evaluation test. The method comprises the following specific steps: after overnight incubation of yersinia pestis and 7 other bacteria and inactivation, bacterial DNA was extracted using QIAamp DNA Mini Kit (Qiagen 51304) and finally eluted with 100 μl TE buffer. The DNA concentration was measured using a Nanodrop instrument, and the concentration was diluted to 0.1 ng/. Mu.L with TE buffer. The obtained DNA was used as a template for a digital PCR reaction system, and the specificity was evaluated by using the multi-target microdroplet digital PCR method of example 2.
The results show that the method for rapidly detecting Yersinia pestis by utilizing the multi-target microdroplet digital PCR established by the invention detects 7 strains of Yersinia pestis near-edge strain DNA such as Yersinia pseudotuberculosis, yersinia enterocolitica, yersinia ruckeri, yersinia keri, yersinia fradiae, yersinia moellensis, yersinia Luo Shiye and the like, and the results are negative and only Yersinia pestis positive. Thus, the method for rapid detection of yersinia pestis based on multi-target microdroplet digital PCR established in example 2 has a good specificity for yersinia pestis.
Example 5 liver tissue sample and soil sample simulation evaluation test
1. Analog sample preparation
200mg of mouse liver tissue is taken, 1mL of physiological saline is added, and the mixture is processed into homogenate by a tissue grinder for standby. Yersinia pestis is cultured overnight, and the culture is washed and resuspended in sterile physiological salineAnd colony counting was performed. Mixing appropriate amount of bacterial liquid with 20mg tissue suspension to obtain tissue bacterial suspension (10) 1 ～10 6 CFU/20mg tissue), i.e., a simulated liver tissue sample, with the tissue suspension without bacteria as a negative control.
Taking environment soil 100mg, adding a proper amount of bacterial liquid 100 mu L to obtain soil samples (10) with different bacterial mixing amounts 1 ～10 6 CFU per 100mg soil), i.e., a simulated soil sample, with non-contaminated soil as a negative control.
2. Simulated sample nucleic acid extraction
Commercial kit for simulating liver tissue samplesBlood and Tissue Kit (Qiagen, 69504) DNA was extracted and eluted with 100. Mu.L TE buffer to obtain liver tissue mimetic sample DNA for storage.
The simulated soil sample was subjected to DNA extraction using a commercial kit soil genomic DNA extraction kit (Tiangen Biochemical technology (Beijing) Co., ltd., DP 336) and eluted with 100. Mu.L TE buffer to obtain a soil simulated sample DNA for storage.
3. Microdroplet digital PCR detection
The detection of liver tissue mimetic and soil mimetic DNA was performed using the method established in example 2 based on the rapid detection of yersinia pestis by multi-target microdroplet digital PCR.
4. Fluorescent PCR detection
The detection of liver tissue simulation sample DNA and soil simulation sample DNA is carried out by adopting a single-target real-time fluorescence PCR detection system, and the reaction system is shown in Table 4. In Table 4, when the primer F is F1, the primer R is R1 and the probe is P1; or when the primer is F2, the primer R is R2, and the probe is P2; or when the primer is F3, the primer R is R3 and the probe is P3.
TABLE 4 Single target real time fluorescent PCR reaction system (20. Mu.L)
Transfer 18. Mu.L into a light-resistant 96-well plate, add 2. Mu.L template into each reaction well, mix well, and place into CFX Opus 96Real-Time PCR System instrument. Reaction conditions: and (3) heat denaturation: 95 ℃ for 5min; annealing/extension (40 cycles): 95 ℃ for 10s;60 ℃,30s (reading step).
Data were analyzed using Bio-Rad CFX Maestro software.
5. Detection result
5.1 liver tissue simulation sample DNA detection results
The multi-target microdroplet digital PCR detection results are shown in (A) - (C) in FIG. 4, and the detection sensitivity of the pestis mixed in the liver tissue sample at different concentrations based on three target genes ypo2088, caf1 and pla is 10 respectively 3 CFU/sample (fig. 4 (a)), 10 3 CFU/sample (fig. 4 (B)), 10CFU/sample (fig. 4 (C)). According to the condition that at least two target genes are required to be positive in pestis PCR detection, the detection sensitivity of the pestis in the liver tissue sample by multi-target microdroplet digital PCR detection is 10 3 CFU/sample。
As shown in FIG. 4 (D-F), for Yersinia pestis of different concentrations incorporated in liver tissue samples, the detection sensitivities based on three target genes ypo2088, caf1, pla were 10 respectively 4 CFU/sample (fig. 4 (D)), 10 4 CFU/sample (FIG. 4 (E), 10CFU/sample (FIG. 4 (F)), the single-target real-time fluorescence PCR detection of pestis in liver tissue has a detection sensitivity of 10 according to the condition that at least two target genes are required to be positive for the pestis PCR detection 4 CFU/sample。
5.2 results of DNA detection of soil simulation samples
As shown in FIGS. 5 (A) - (C), the detection sensitivity of pestis with different concentrations incorporated in soil sample based on three target genes ypo2088, caf1, and pla is 10 3 CFU/sample (fig. 5 (a)), 10 2 CFU/sample (fig. 5 (B)), 10CFU/sample (fig. 5 (C)). According to the condition that at least two target genes are required to be positive in pestilence PCR detection, multi-target microdroplet digital PCR detection of pestilence in soil samplesHas a detection sensitivity of 10 2 CFU/sample。
As shown in (D) - (F) of FIG. 5, the detection sensitivity of Yersinia pestis of different concentrations incorporated in soil samples based on three target genes ypo2088, caf1, pla was 10 respectively 4 CFU/sample (fig. 5 (D)), 10 4 CFU/sample (fig. 5 (E)), 10CFU/sample (fig. 5 (F)). According to the condition that at least two target genes are required to be positive in pestis PCR detection, the detection sensitivity of single-target real-time fluorescent PCR detection of pestis in soil is 10 4 CFU/sample。
In conclusion, aiming at the bacteria-doped liver tissue sample, the detection sensitivity of the multi-target microdroplet digital PCR is 10 times better than that of the single-target real-time fluorescence PCR; aiming at the bacteria-doped soil sample, the detection sensitivity of the multi-target microdroplet digital PCR is 100 times better than that of the single-target real-time fluorescence PCR; the method for detecting the Yersinia pestis multi-target microdroplet digital PCR has obvious detection sensitivity advantage compared with the traditional real-time fluorescent PCR method.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.
Claims (10)
1. A composition based on microdroplet digital PCR detection or assisted detection of Yersinia pestis (Yersinia pestis), consisting of a multiplex PCR primer pair composition and a probe composition; the multiplex PCR primer pair consists of a primer pair A, a primer pair B and a primer pair C, and the probe composition consists of three probes P1, P2 and P3; labeling a fluorescent group on the 5 'terminal nucleotide of the nucleotide sequence of each of the three probes, and labeling a quenching group on the 3' terminal nucleotide of the nucleotide sequence of each probe; the primer pair A can be specifically combined with a target gene ypo2088 on a yersinia pestis genome chromosome, the primer pair B can be specifically combined with a yersinia pestis genome plasmid target gene pla, and the primer pair C can be specifically combined with a yersinia pestis genome plasmid target gene caf1; the 5' -end nucleotide of the P1 and the P2 is marked with the same fluorescent group, and the P3 and the P1 and the P2 are marked with different fluorescent groups.
2. The composition of claim 1, wherein: the molar ratio of P1 to P2 is more than or equal to 2:1.
3. composition according to claim 1 or 2, characterized in that: the P1 can be specifically bound to a target gene ypo2088 on the genome chromosome of Yersinia pestis, and the final concentrations of P1, P2 and P3 in the multiplex PCR system are 500nM, 250nM and 250nM respectively.
4. A composition according to any one of claims 1 to 3, characterized in that: the primer pair A is a primer pair consisting of single-stranded DNA shown in a sequence 1 and single-stranded DNA shown in a sequence 2 in a sequence table; the primer pair B is a primer pair consisting of single-stranded DNA shown in a sequence 3 and single-stranded DNA shown in a sequence 4 in a sequence table; the primer pair C is a primer pair consisting of single-stranded DNA shown in a sequence 5 and single-stranded DNA shown in a sequence 6 in a sequence table; the nucleotide sequence of the probe P1 is a sequence 3 in a sequence table; the nucleotide sequence of the probe P2 is a sequence 6 in a sequence table; the nucleotide sequence of the probe P3 is a sequence 9 in a sequence table.
5. A reagent and/or kit for the identification or assisted identification of Yersinia pestis (Yersinia pestis), characterized in that: the reagent and/or kit contains the composition of any one of claims 1-4.
6. A system for identifying or aiding in the identification of Yersinia pestis (Yersinia pestis), characterized in that: the system comprises the reagent and/or kit according to claim 5.
7. The system according to claim 6, wherein: the system also contains a digital PCR system.
8. A method for detecting or aiding in the detection of Yersinia pestis (Yersinia pestis), characterized by: the method comprises performing a microdroplet digital PCR analysis on a test sample using the composition of any one of claims 1-4, or the reagent or kit of claim 5, or the system of claim 6 or 7, and determining or aiding in determining whether the test sample is or contains yersinia pestis or is infected with yersinia pestis based on the microdroplet digital PCR detection results.
9. The method according to claim 8, wherein: the molar ratio of P1 to P2 in the microdroplet digital PCR system is greater than or equal to 2:1.
10. use of a composition according to any one of claims 1 to 4 for the preparation of a product for the detection or co-detection of yersinia pestis.
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