CN118979116A - Anthrax, plague and brucellosis triple RPA detection kit, primer probe combination and application - Google Patents
Anthrax, plague and brucellosis triple RPA detection kit, primer probe combination and application Download PDFInfo
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Abstract
The invention discloses an anthrax, plague and bubbly triple RPA detection kit, a primer probe composition and application. The invention discloses LFD-RPA-based bacillus anthracis, plague bacillus and brucella detection kit and a primer probe composition for detecting bacillus anthracis, a primer probe composition for detecting plague bacillus, a primer probe composition for detecting brucella, and RPA-based bacillus anthracis, plague bacillus and brucella detection kit and a primer probe composition for detecting bacillus anthracis, a primer probe composition for detecting plague bacillus and a primer probe composition for detecting brucella. The detection method provided by the invention can detect bacillus anthracis, plague bacillus and brucella simultaneously, does not need to depend on professionals, can be used for on-site detection, and has the characteristics of simplicity, convenience, rapidness, sensitivity and specificity.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an anthrax, plague and bubber triple RPA detection kit, a primer probe composition and application.
Background
Bacillus anthracis (Bacillus anthracis), plague (YERSINIA PESTIS) and Brucella (Brucella) can cause severe anthrax, plague and brucellosis in humans and animals, severely threatening public health. Herbivores generally die quickly when they eat anthrax spores in the soil during grass feeding, and humans often eat or contact these dead animals and become infected with anthrax; plague is transmitted through the bite of rat fleas, and has high infection speed, strong transmissibility, fast morbidity and high mortality; brucellosis is often infected by direct contact with infected animals, eating or drinking contaminated animal products, or inhalation of airborne pathogens. Early detection and diagnosis of the three pathogens are of great importance for disease spread and control.
Molecular biological detection methods based on polymerase chain reaction (polymerase chain reaction, PCR), real-time fluorescent quantitative PCR (Quantitative Real-time PCR, qPCR) and the like have been widely used for detecting pathogenic bacteria, and although these methods can accurately detect pathogenic bacteria, they often take a long time, and depending on precise instruments (such as qPCR instrument) and professional biotechnologies, it is basically impossible to realize field detection. As a novel nucleic acid isothermal amplification technology, the recombinase polymerase amplification (Recombinase Polymerase Amplification, RPA) can realize rapid detection of a target to be detected within 10-30 min at the optimal temperature of 37-42 ℃, has the advantages of high sensitivity, strong specificity, low instrument dependence, capability of integrating multiple detection modes and the like, and is particularly suitable for real-time detection of a base layer and a site. The lateral flow test strip (lateral flow dipsticks, LFDs) technology is a classical bedside detection method based on antigen and antibody immune reaction, and has the advantages of rapidness, simplicity, low cost and the like. The RPA and the LFD are combined, so that the visual detection of the amplified product can be realized, complex instruments and equipment are not needed, the method is suitable for on-site rapid detection, and the method has extremely wide application prospect.
Based on LFD-RPA technology, how to realize the specificity and sensitivity detection of three pathogenic bacteria of bacillus anthracis, plague bacillus and brucellosis simultaneously is one of the hot spots focused by the technicians in the field.
Disclosure of Invention
The invention provides a kit, a primer probe composition and application for detecting bacillus anthracis, plague bacillus and brucellosis based on LFD-RPA.
The invention also provides a kit for detecting bacillus anthracis, plague bacillus and brucellosis bacillus based on RPA, a primer probe composition and application.
In a first aspect, the invention provides a kit for detecting bacillus anthracis, plague bacillus and brucella, comprising a composition for detecting bacillus anthracis, a composition for detecting plague bacillus, a composition for detecting brucella and lateral flow test paper;
The composition for detecting bacillus anthracis comprises a primer lefRPA-F, a primer lefRPA-R-1 and a Probe lefRPA-Probe-1; the composition for detecting the plague bacillus comprises a primer YP01-3F, a primer YP01-3R-1 and a probe YP01-3Prboe-1; the composition for detecting Brucella comprises a primer bscp RPA-F, a primer bscp RPA-R-1 and a Probe bscp RPA-Probe-1;
The structural general formulas of the Probe lefRPA-Probe-1, the Probe YP01-3Prboe-1 and the Probe bscp RPA-Probe-1 are shown in the formula 1:
5'-X- (n) a-H- (n) b-Y-3' formula 1;
in formula 1, X is a second labeling group, (n) a is a first single-stranded DNA molecule, H is a tetrahydrofuran residue, (n) b is a second single-stranded DNA molecule, and Y is a blocking group;
The primer lefRPA-F is single-stranded DNA with a nucleotide sequence shown as SEQ ID NO. 1, and the primer lefRPA-R-1 is single-stranded DNA with a nucleotide sequence shown as SEQ ID NO. 2 modified by a first marker group; in the Probe lefRPA-Probe-1, (n) a is a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO. 3, and (n) b is a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO. 4;
The primer YP01-3F is single-stranded DNA with a nucleotide sequence shown as SEQ ID NO. 5, and the primer YP01-3R-1 is single-stranded DNA with a nucleotide sequence shown as SEQ ID NO. 6 modified by a first marker group; in the probe YP01-3Prboe-1, (n) a is a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO. 7, and (n) b is a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO. 8;
The primer bscp RPA-F is single-stranded DNA with a nucleotide sequence shown as SEQ ID NO. 9, the primer bscp RPA-R-1 is single-stranded DNA with a first marker group modified nucleotide sequence shown as SEQ ID NO. 10, and in the Probe bscp RPA-Probe-1, (n) a is single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO. 11, and (n) b is single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO. 12.
In the kit, the primer lefRPA-F is a forward primer, the primer lefRPA-R-1 is a reverse primer, and the nucleotide at the 1 st position of the 5' end of the primer lefRPA-R-1 is marked by a first marking group.
In the kit, the primer YP01-3F is a forward primer, the primer YP01-3R-1 is a reverse primer, and the 1 st nucleotide at the 5' end of the primer YP01-3R-1 is marked by a first marking group.
In the kit, the primer bscp RPA-F is a forward primer, the primer bscp RPA-R-1 is a reverse primer, and the 1 st nucleotide at the 5' end of the primer bscp RPA-R-1 is marked by a first marking group.
Further, the first labeling group is a small molecule compound such as biotin or a fluorescent group which can be recognized by a monoclonal antibody. Furthermore, the 5' -terminal nucleotide 1 of each of the primers lefRPA-R-1, YP01-3R-1 and bscp RPA-R-1 was labeled with biotin.
The structural general formulas of the Probe lefRPA-Probe-1, the Probe YP01-3Prboe-1 and the Probe bscp31RPA-Probe-1 are shown in the formula 1:
5'-X- (n) a-H- (n) b-Y-3' formula 1;
In formula 1, X is a second labeling group, (n) a is a first single-stranded DNA molecule, H is a tetrahydrofuran residue, (n) b is a second single-stranded DNA molecule, and Y is a blocking group.
Further, the second labeling groups X labeled at the 5' ends of the probes lefRPA-Probe-1, the probes YP01-3Prboe-1 and the probes bscp31RPA-Probe-1 are different so as to bind with different antibodies coated on the lateral flow test paper detection area. The second labeling groups marked at the 5' end of the Probe lefRPA-Probe-1, the Probe YP01-3Prboe-1 and the Probe bscp RPA-Probe-1 independently select small molecular compounds such as fluorescent groups, digoxin and the like which can be recognized by monoclonal antibodies.
Further, the fluorescent group is selected from one or more of FITC, TAMRA, FAM, HEX, CY, CY5, ROX, texas Red.
Further, the 5' end of the Probe lefRPA-Probe-1 is marked with a fluorescent group FITC; the 5' end of the probe YP01-3Prboe-1 is marked with digoxin DIG; the 5' end of the Probe bscp RPA-Probe-1 is marked with a fluorescent group TAMRA.
Further, the blocking group is a group having the purpose of blocking nucleic acid extension. Still further, the blocking groups are independently selected from one or more of C3 Spacer, C6 Spacer, C9 Spacer. Further, the 3' ends of the probes lefRPA-Probe-1, YP01-3Prboe-1 and bscp RPA-Probe-1 are marked with C3 Spacer.
The concentration of the primer lefRPA-F, the primer lefRPA-R-1, the Probe lefRPA-Probe-1, the primer YP01-3F, the primer YP01-3R-1, the Probe YP01-3Prboe-1, the primer bscp RPA-F, the primer bscp31RPA-R-1 and the Probe bscp31RPA-Probe-1 is 10 mu mol/L to 100 mu mol/L.
According to the kit, the transverse flow test paper sequentially comprises the sample pad, the binding pad, the detection area and the water absorption pad along the liquid flow direction, wherein the binding pad is attached with the colloidal gold marker, and the colloidal gold marker is a combination body obtained by combining the colloidal gold with the substance specifically combined with the first marker group. Further, when the first labeling group is biotin, the substance specifically bound to the first labeling group is an anti-biotin monoclonal antibody.
The detection area comprises an bacillus anthracis detection line, a plague bacillus detection line, a brucellosis detection line and a quality control line, wherein antibodies resisting a second marker group are respectively coated on the bacillus anthracis detection line, the plague bacillus detection line and the brucellosis detection line; specifically, when a second labeling group labeled on a Probe lefRPA-Probe-1 for detecting bacillus anthracis is FITC, the bacillus anthracis detection line is coated with an anti-FITC antibody, and the anti-FITC antibody is named as anti-FITC; when the second labeling group labeled on the probe YP01-3Prboe-1 for detecting plague bacillus is DIG, the bacillus anthracis detection line is coated with an anti-DIG antibody, and the anti-DIG is named as anti-DIG; when the second labeling group labeled on the RPA-Probe-1 of the Probe bscp for detecting Brucella is TARMA, the Brucella detection line is coated with an antibody against TARMA and is named as anti-TARMA.
Further, the lateral flow test strip may be a strip, i.e., a lateral flow test strip.
The kit as described above further comprises enzymes required for the RPA reaction, including recombinases, single-stranded DNA binding proteins and strand displacement DNA polymerases.
In a second aspect, the invention provides a kit for detecting bacillus anthracis, plague bacillus and brucella, comprising a composition for detecting bacillus anthracis, a composition for detecting plague bacillus and a composition for detecting brucella;
The composition for detecting bacillus anthracis comprises a primer lefRPA-F, a primer lefRPA-R and a Probe lefRPA-Probe; the composition for detecting the plague bacillus comprises a primer YP01-3F, a primer YP01-3R and a probe YP01-3Prboe; the composition for detecting Brucella comprises a primer bscp RPA-F, a primer bscp RPA-R and a Probe bscp RPA-Probe;
The structure of the probe is shown in formula 2:
5'- (n) c-H- (n) d-Y-3' formula 2;
in formula 2, (n) c is a first single-stranded DNA molecule modified with a fluorescent group, H is a tetrahydrofuran residue, (n) d is a second single-stranded DNA molecule modified with a quenching group, Y is a blocking group;
The primer lefRPA-F is single-stranded DNA with a nucleotide sequence shown as SEQ ID NO. 1, the primer lefRPA-R is single-stranded DNA with a nucleotide sequence shown as SEQ ID NO. 2, and in the Probe lefRPA-Probe, (n) c is a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO. 3 modified by using a fluorescent group, and (n) d is a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO. 4 modified by using a quenching group;
The primer YP01-3F is single-stranded DNA with a nucleotide sequence shown as SEQ ID NO. 5, the primer YP01-3R is single-stranded DNA with a nucleotide sequence shown as SEQ ID NO. 6, the probe YP01-3Prboe is (n) c is a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO. 7 modified by using a fluorescent group, and (n) d is a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO. 8 modified by using a quenching group;
the primer bscp RPA-F is single-stranded DNA with a nucleotide sequence shown as SEQ ID NO. 9, the primer bscp RPA-R is single-stranded DNA with a nucleotide sequence shown as SEQ ID NO. 10, the Probe bscp RPA-Probe is composed of (n) c which is a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO. 11 modified by using a fluorescent group and (n) d which is a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO. 12 modified by using a quenching group.
The kit as described above, wherein the fluorophores are independently selected from at least one of FAM, VIC, HEX, TRT, CY, CY5, ROX, JOE, FITC, TET, NED, TAMRA, LC RED460, LC RED705, quasar705, texas RED; the quenching group is independently selected from one of BHQ1, BHQ2, BHQ3, MGB and Eclipse.
In the kit described above, the fluorescent group is modified at the 1 st nucleotide or the 2 nd nucleotide at the 3 'end of the first single-stranded DNA molecule, the quenching group is modified at the 1 st nucleotide or the 2 nd nucleotide at the 5' end of the second single-stranded DNA molecule, and the first single-stranded DNA molecule and the second single-stranded DNA molecule are linked by a tetrahydrofuran residue.
Further, in the Probe lefRPA-Probe, a fluorescent group FAM is modified on the 2 nd nucleotide of the 3' end of a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO. 3, a quenching group BHQ1 is modified on the 2 nd nucleotide of the 5' end of the single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO. 4, and a blocking group C3-spacer is modified on the 1 st nucleotide of the 3' end; and the 1 st nucleotide of the 3 'end of the single-stranded DNA shown in SEQ ID NO. 3 modified by the fluorescent group FAM and the 1 st nucleotide of the 5' end of the single-stranded DNA shown in SEQ ID NO. 4 modified by the quenching group BHQ1 are connected through tetrahydrofuran residue.
Further, in the probe YP01-3Prboe, a fluorescent group HEX is modified on the 2 nd nucleotide of the 3' end of a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO. 7, a quenching group BHQ1 is modified on the 2 nd nucleotide of the 5' end of the single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO. 8, and a blocking group C3-spacer is modified on the 1 st nucleotide of the 3' end; and the 1 st nucleotide of the 3 'end of the single-stranded DNA shown in SEQ ID NO. 7 modified by the fluorescent group HEX and the 1 st nucleotide of the 5' end of the single-stranded DNA shown in SEQ ID NO. 8 modified by the quenching group BHQ1 are connected through tetrahydrofuran residue.
Further, in the Probe bscp RPA-Probe, a fluorescent group Texas Red is modified on the 1 st nucleotide of the 3' end of a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO. 11, a quenching group BHQ2 is modified on the 1 st nucleotide of the 5' end of the single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO. 12, and a blocking group C3-spacer is modified on the 1 st nucleotide of the 3' end; and the 1 st nucleotide of the 3 'end of the single-stranded DNA shown in SEQ ID NO. 11 modified by the fluorescent group Texas Red and the 1 st nucleotide of the 5' end of the single-stranded DNA shown in SEQ ID NO. 12 modified by the quenching group BHQ2 are connected through tetrahydrofuran residue.
In a third aspect, the present invention provides a composition comprising at least one of the composition for detecting bacillus anthracis, the composition for detecting plague bacillus and the composition for detecting brucellosis in the kit provided in the first aspect of the present invention.
In a fourth aspect, the present invention provides a composition comprising at least one of the composition for detecting bacillus anthracis, the composition for detecting plague bacillus and the composition for detecting brucellosis in the kit provided in the second aspect of the present invention.
In a fifth aspect, the invention provides the use of a kit and/or composition as described in any one of the preceding aspects:
a1 Detection or auxiliary detection of pathogenic bacteria;
a2 Preparing a product for detecting or assisting in detecting pathogenic bacteria;
the pathogenic bacteria are at least one of bacillus anthracis, plague bacillus and brucellosis.
It will be appreciated that the kit provided in the first aspect of the invention may be used when detecting only one or two of bacillus anthracis, plague bacillus and brucella, and that primer probe compositions for detecting one or two of bacillus anthracis, plague bacillus and brucella may be used, without further limitation.
In a sixth aspect, the present invention provides a method of detecting a pathogen comprising:
b1 Extracting genome DNA of a sample to be detected;
b2 Using the genomic DNA as a template, performing LFD-RPA detection with the composition provided in the third aspect of the present invention or the kit provided in the first aspect; determining whether a sample to be tested contains the pathogenic bacteria according to the strip in the transverse flow test paper;
the pathogenic bacteria are at least one of bacillus anthracis, plague bacillus and brucellosis.
The method for determining whether the sample to be tested contains at least one of bacillus anthracis, plague bacillus and brucella according to the strip in the transverse flow test paper comprises the following steps: the detection area of the transverse flow test paper (LFD test paper for short) comprises an bacillus anthracis detection line, a plague bacillus detection line, a Brucella detection line and a quality control line; if the quality control line of the LFD test paper has a red band and at least one of the three detection lines has a red band, the result is positive, which indicates that the sample to be tested contains at least one pathogenic bacterium of bacillus anthracis, plague bacillus and brucellosis; if the LFD test paper only has one red band positioned on the quality control line, the result is negative, which indicates that the sample to be tested does not contain any one of bacillus anthracis, plague bacillus and brucella; if the LDF test paper does not have any red bands, the detection result is invalid.
The method as described above, step B2) the total volume of the reaction system detected by LFD-RPA was 50. Mu.L: comprises 29.5. Mu.L of buffer, 1.1. Mu.L of primer lefRPA-F, 1.1. Mu.L of primer lefRPA-R-1, 1.1. Mu.L of primer YP01-3F, 1.1. Mu.L of primer YP01-3R-1, 1.1. Mu.L of primer bscp RPA-F, 1.1. Mu.L of primer bscp RPA-R-1, 0.1. Mu.L of Probe lefRPA-Probe-1, 0.1. Mu.L of Probe YP01-3RPA-Probe-1, 0.4. Mu.L of Probe bscp RPA-Probe-1 (the concentrations of the above six primers and the three probes are 10. Mu.mol/L), 7.8. Mu.L of DEPC water, 3. Mu.L of genomic DNA of the sample to be tested, and 2.5. Mu.L of 280 mmol/L of MgOAc solution.
In the method, the reaction system is added into RPA freeze-dried enzyme powder to carry out RPA amplification reaction, and after the reaction is finished, the amplification product is dripped onto LFD test paper to carry out detection. Further, the temperature of the RPA amplification reaction is 39 ℃, the reaction time is 10 minutes, and after the amplified product is dripped on LFD test paper, the reaction is observed after the reaction is placed at room temperature for 10 minutes.
In a seventh aspect, the present invention provides a method of detecting a pathogen, comprising:
c1 Extracting genome DNA of a sample to be detected;
C2 Using the genomic DNA as a template, performing RPA detection with the composition provided in the fourth aspect of the present invention or the kit provided in the second aspect; determining whether the sample to be detected contains the pathogenic bacteria according to the fluorescence signal intensity;
the pathogenic bacteria are at least one of bacillus anthracis, plague bacillus and brucellosis.
The detection methods provided in the sixth and seventh aspects of the present invention are methods for the purpose of non-disease diagnosis for detecting the presence or absence of pathogenic bacteria in a sample.
The sample to be tested provided in the sixth and seventh aspects of the present invention may be an environmental sample, a clinical sample, an article, or the like.
The invention provides a kit, a primer probe composition and a method for detecting bacillus anthracis, plague bacillus and brucellosis based on LFD-RPA, which are simple, quick, sensitive and specific, can specifically detect any one of bacillus anthracis, plague bacillus and brucellosis from different pathogenic bacteria, do not need large-scale instrument and equipment and professional technicians, and are suitable for finishing the detection of the pathogenic bacteria in a sample on the base site.
The invention provides a reagent kit, a primer probe composition and a method for detecting bacillus anthracis, plague bacillus and brucellosis based on RPA, which have the characteristics of good specificity and high sensitivity, and can detect bacillus anthracis, plague bacillus and brucellosis simultaneously in one reaction tube without cross reaction.
The invention applies two detection kits to the newly separated strain, clinical blood sample and simulated sample, and experimental results show that the two detection kits have good effects in clinical blood sample, separated strain and simulated sample.
Drawings
FIG. 1 is the RPA-specific detection results of primer probe compositions for detection of Bacillus anthracis, phytophthora, and Brucella; wherein A is the result of performing single-weight RPA detection on seven pathogenic bacteria DNA samples by using a primer probe composition for detecting bacillus anthracis, B is the result of performing single-weight RPA detection on seven pathogenic bacteria DNA samples by using a primer probe composition for detecting bacillus plague, and C is the result of performing single-weight RPA detection on seven pathogenic bacteria DNA samples by using a primer probe composition of brucella;
FIG. 2 shows the RPA sensitivity detection results of primer probe compositions for detection of Bacillus anthracis, phytophthora, and Brucella; wherein A is the single RPA detection result of the primer probe composition for detecting bacillus anthracis on the lef genes of bacillus anthracis with different concentrations, B is the single RPA detection result of the primer probe composition for detecting bacillus plague on the YP01-3 genes of bacillus plague with different concentrations, C is the single RPA detection result of the primer probe composition for detecting bacillus brucei on the bscp genes of bacillus brucei; ns is not significant, and P is less than or equal to 0.05;
FIG. 3 is a multiplex RPA detection of primer probe compositions for detection of B.anthracis, B.plague and B.brucei; wherein, A is the dual RPA detection result of the primer probe composition for detecting bacillus anthracis and plague bacillus on four pathogenic bacteria combinations and two pathogenic bacteria, B is the dual RPA detection result of the primer probe composition for detecting bacillus plague and brucellosis on four pathogenic bacteria combinations and two pathogenic bacteria, C is the dual RPA detection result of the primer probe composition for detecting bacillus anthracis and brucellosis on four pathogenic bacteria combinations and two pathogenic bacteria, D is the triple RPA detection result of the primer probe composition for detecting bacillus anthracis, plague bacillus and brucellosis on eight pathogenic bacteria combinations;
FIG. 4 shows the LFD-RPA detection results of primer probe compositions for detection of Bacillus anthracis, phytophthora, and Brucella; wherein A is the detection result of a primer probe composition for detecting bacillus anthracis and plague bacillus on pathogenic bacteria, B is the detection result of a primer probe composition for detecting bacillus brucei and plague bacillus on pathogenic bacteria, C is the detection result of a primer probe composition for detecting bacillus anthracis and plague bacillus on pathogenic bacteria, and D is the detection result of a primer probe composition for detecting bacillus anthracis, plague bacillus and plague bacillus on pathogenic bacteria;
FIG. 5 shows the results of the detection of RPA and LFD-RPA on clinical samples, isolates, simulated samples with primer probe compositions for detection of Bacillus anthracis, phytophthora, and Brucella; wherein A is the RPA detection result and LFD-RPA detection result of the primer probe composition for detecting bacillus anthracis, plague bacillus and brucella on clinical blood samples with the numbers of S1-S19, B is the PCR detection result of the clinical blood samples with the numbers of S1-S19, C is the RPA detection result and LFD-RPA detection result of the primer probe composition for detecting bacillus anthracis, plague bacillus and brucella on bacillus anthracis Xa01, D is the PCR detection result of bacillus anthracis Xa01, E is the RPA detection result and LFD-RPA detection result of the primer probe composition for detecting bacillus anthracis, plague bacillus and brucella on plague simulated blood genome samples, and F is the PCR detection result of the plague simulated blood genome samples.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions thereof will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments, which should not be construed as limiting the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. In the description of the present invention, it is to be understood that the terminology used is for the purpose of description only and is not to be interpreted as indicating or implying relative importance.
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.
The test strains and sources thereof are shown in Table 1, wherein CICC is China center for type culture Collection of Industrial microorganisms, and the website is http:// www.china-CICC. The Yersinia pestis sample used is Yersinia pestis EV76 genome, which is stored in the laboratory.
TABLE 1 test strain information
The test strains of Table 1 were obtained from the military medical institute of the national institute of civil release, military, and could not be used for other purposes.
EXAMPLE 1 RPA detection and LFD-RPA detection
1. Preparation of bacterial genome templates
The test strains shown in Table 1 were cultured in a solid medium, single colonies were picked up and cultured in a liquid medium with shaking, bacterial liquid was collected after overnight culture, DNA was extracted by using a bacterial genomic DNA extraction kit (TIANGEN DP, purchased from Tiangen Biochemical technologies (Beijing) Co., ltd.) after inactivation of the bacterial liquid, and finally DNA was eluted with 60. Mu.L of TE buffer, and the obtained genomic nucleic acid was quantified by a NanoDrop 2000 spectrophotometer to obtain a genomic template.
2. Design and Synthesis of primers and probes
Based on the RPA method, forward primer lefRPA-F, reverse primer lefRPA-R and Probe lefRPA-Probe are designed according to the lef gene on bacillus anthracis plasmid pXO 1. The plat gene on pPCP plasmid was selected according to pesticium, and the forward primer YP01-3F, the reverse primer YP01-3R and the probe YP01-3Prboe were designed. The forward primer bscp RPA-F, the reverse primer bscp RPA-R and the Probe bscp RPA-Probe were designed based on bscp gene of Brucella.
Based on the LFD-RPA method, a forward primer lefRPA-F, a reverse primer lefRPA-R-1 and a Probe lefRPA-Probe-1 for detecting bacillus anthracis are designed. The forward primer YP01-3F, the reverse primer YP01-3R-1 and the probe YP01-3Prboe-1 are used for detecting the plague bacillus. Forward primer bscp RPA-F, reverse primer bscp RPA-R-1 and Probe bscp RPA-Probe-1 for detecting Brucella.
The nucleotide sequences of the above primers and probes are specifically shown in Table 2.
TABLE 2 primer and probe sequences
The primer and the probe are synthesized by Jiangsu Saixue aerobe technology Co., ltd and Anhui general biological Co., ltd, and both the primer and the probe are purified by HPLC.
In Table 2, 6-FAM is 6-carboxyfluorescein (6-Carboxyfluorescein); biotin is Biotin; FITC is fluorescein isothiocyanate (Fluorescein Isothiocyanate); dig is digoxin (Digoxin); TAMRA is 6-TAMRA (6-carboxytetramethyl rhodamine ); HEX is 6-HEX (6-hexachloro-6-methylfluorescein, 6-Hexachlorofluorescein); texas Red is Texas Red, also known as Sulforhodamine 101 (Sulfohodamine 101); BHQ1 and BHQ2 are quenching groups.
3. RPA and LFD-RPA detection systems and methods
The RPA fluorescence detection method comprises the following steps: extracting DNA of a sample to be detected, taking the DNA of the sample to be detected as a template, adding an RPA detection system with the total volume of 50 mu L into an RPA freeze-dried enzyme powder tube (purchased from TwistDx company and with the product number of TABAS KIT), performing nucleic acid amplification reaction at 39 ℃, performing fluorescence signal detection on an amplification product after reaction of 20 min, and determining whether the sample to be detected contains at least one of bacillus anthracis, plague bacillus and brucellosis. And if at least one fluorescent signal of FAM, HEX and Texas Red appears in the sample to be detected, indicating that the sample to be detected contains corresponding pathogenic bacteria. If the detection result has no fluorescent signal, the sample to be detected does not contain any one of bacillus anthracis, plague bacillus and brucellosis.
Wherein, the RPA detection system includes: 29.5. Mu.L of buffer, 1.1. Mu.L of primer lefRPA-F solution, 1.1. Mu.L of primer lefRPA-R solution, 1.1. Mu.L of primer YP01-3F solution, 1.1. Mu.L of primer YP01-3R solution, 1.1. Mu.L of primer bscp RPA-F solution, 1.1. Mu.L of primer bscp RPA-R solution, 0.3. Mu.L of Probe lefRPA-Probe solution, 0.3. Mu.L of Probe YP01-3Prboe solution, 0.3. Mu.L of Probe bscp RPA-Probe solution (the concentrations of the above six primer solutions and the three Probe solutions are 10. Mu.mol/L), 7.5. Mu.L of DEPC water, 3. Mu.L of nucleic acid template, 2.5. Mu.L of 280 mmol/L of MgOAc solution.
3.2 LFD-RPA detection
3.2.1 LFD test strips were purchased from Nanjing's tripod Biotechnology Co., ltd. Under the product number AJG10-50.
3.2.2, LFD-RPA detection method comprises: extracting DNA of a sample to be detected, taking the DNA of the sample to be detected as a template, adding RPA freeze-dried enzyme powder and an LFD-RPA detection system, performing nucleic acid amplification reaction at 39 ℃ for 10min, placing an amplification product on an LFD test strip, standing at room temperature for 10min, and observing. And determining whether the sample to be tested contains at least one of bacillus anthracis, plague bacillus and brucella according to the LFD test strip. If the quality control line and the corresponding detection line of the LFD test strip are both red stripes, the result is positive, which indicates that the sample to be tested contains corresponding pathogenic bacteria; if the LFD test strip only has a red strip of a quality control line, the result is negative, which indicates that the sample to be tested does not contain any one of bacillus anthracis, plague bacillus and brucella; and if the quality control line does not have a red stripe, indicating that the detection result is invalid.
The LFD-RPA detection system comprises: 29.5. Mu.L of buffer, 1.1. Mu.L of primer lefRPA-F, 1.1. Mu.L of primer lefRPA-R-1, 1.1. Mu.L of primer YP01-3F, 1.1. Mu.L of primer YP01-3R-1, 1.1. Mu.L of primer bscp RPA-F, 1.1. Mu.L of primer bscp RPA-R-1, 0.1. Mu.L of Probe lefRPA-Probe-1, 0.1. Mu.L of Probe YP01-3RPA-Probe-1, 0.4. Mu.L of Probe bscp RPA-Probe-1 (the concentrations of the above six primers and the three probes are 10. Mu.mol/L), 7.8. Mu.L of DEPC water, 3. Mu.L of nucleic acid template, 2.5. Mu.L of 280 mmol/L of MgOAc solution.
Example 2 assay Performance analysis
1. Specificity analysis
The primers designed in Table 2 were subjected to specificity verification analysis using genomic DNA of the strain to be tested shown in Table 1 as a template, nuclease-free water as a blank, and Bacillus anthracis, pestilence and Brucella as positive samples, respectively.
The specific experimental steps are as follows: selecting genome DNA of a strain to be tested as a template, and respectively carrying out real-time fluorescence RPA reaction with the primer probe compositions corresponding to the primer probe compositions in the table 2, wherein an RPA reaction system comprises: 29.5. Mu.L of reconstitution buffer, 2.1. Mu.L of forward primer (10. Mu. Mol/L), 2.1. Mu.L of reverse primer (10. Mu. Mol/L), 0.6. Mu.L of probe (10. Mu. Mol/L), 11.2. Mu.L of DEPC water and 2. Mu.L of nucleic acid template, adding RPA freeze-dried enzyme powder, shaking uniformly, centrifuging briefly, adding 2.5. Mu.L of 280 mmol/L of MgOAc solution, shaking uniformly, and centrifuging briefly. And (3) placing the sample into a real-time fluorescence quantitative PCR instrument, detecting a fluorescence signal according to a software operation instruction, and setting three repeated experiments. The detection results are shown in FIG. 1.
As shown in FIG. 1, bacillus anthracis can be distinguished from other pathogens by primers lefRPA-F, lefRPA-R and probes lefRPA-probes; the pestis can be distinguished from other pathogenic bacteria by the primer YP01-3RPA-F, YP01-3RPA-R and the Probe YP01-3 RPA-Probe; brucella can be distinguished from other pathogenic bacteria by primer bscp RPA-F, bscp RPA-R and Probe bscp RPA-Probe. The specificity and stability of the primer probe provided in Table 2 are better, and the fluorescence value gradually becomes stable at 10 minutes in the detection reaction.
2. Sensitivity analysis
To verify the sensitivity of the RPA reaction, genomic DNA was gradually diluted from 10 5 copies/. Mu.l to 10 -1 copies/. Mu.l in a tenfold gradient. The diluted genome DNA is used as a template, nuclease-free water is used as a blank control, the real-time fluorescence RPA reaction is carried out, the experiment is repeated for 3 times, the statistical analysis is carried out on the experimental data, and the analysis result is shown in figure 2.
As shown in FIG. 2, the higher the template copy number, the stronger the fluorescence signal, wherein the combined sensitivity of the lef and bscp31 primer probes can reach 10 1 copies/. Mu.L, and the combined sensitivity of the YP01-3 primer probes can reach 10 0 copies/. Mu.L.
3. Multiplex RPA reactions
The genome DNA of two different pathogenic bacteria is used as a template to carry out real-time fluorescence RPA reaction with RPA primers to carry out double RPA detection, and a double RPA detection reaction system (50 mu L) comprises 29.5 mu L of buffer solution, 2X 1.1 mu L of forward primer, 2X 1.1 mu L of reverse primer, 2X 0.3 mu L of probe (the concentration of the primer and the probe is 10 mu mol/L), 10 mu L of DEPC water, 3 mu L of nucleic acid template and 2.5 mu L of MgOAc solution of 280 mmol/L. The detection results are shown as A-C in FIG. 3.
The genome of three different pathogenic bacteria is used as a template, real-time fluorescence RPA reaction with RPA primer is carried out, triple RPA detection is carried out, and a triple RPA detection reaction system (50. Mu.L) comprises 29.5. Mu.L buffer solution, 1.1. Mu.L primer lefRPA-F, 1.1. Mu.L primer lefRPA-R, 1.1. Mu.L primer YP01-3F, 1.1. Mu.L primer YP01-3R, 1.1. Mu.L primer bscp RPA-F, 1.1. Mu.L primer bscp RPA-R, 0.3. Mu.L Probe lefRPA-Probe, 0.3. Mu.L Probe YP01-3Prboe, 0.3. Mu.L Probe bscp RPA-Probe (the concentrations of the above six primers and the three probes are 10. Mu.mol/L), 7.5. Mu.L DEPC water, 3. Mu.L nucleic acid template, and 2.5. Mu.L MgOAc solution of mmol/L. The detection result is shown as D in fig. 3.
As can be seen from FIG. 3, the double RPA reaction and the triple RPA reaction have good feasibility, and 3 pathogenic bacteria can be detected simultaneously in one reaction system without cross reaction.
4. LFD-RPA reaction
Primer probes for detecting bacillus anthracis use a primer lefRPA-F, a primer lefRPA-R-1 and a primer lefRPA-Probe-1, primer YP01-3F, primer YP01-3R-1 and a Probe YP01-3Prboe-1 for detecting bacillus anthracis, primer bscp RPA-F, primer bscp RPA-R-1 and Probe bscp31RPA-Probe-1 for detecting bacillus anthracis, the amount of the primer and other reagents in a triple RPA fluorescence method is adopted, and the Probe proportion is lefRPA-Probe-1 and 0.1 mu L; bscp31RPA-Probe-1,0.4. Mu.L; YP01-3Prboe-1, 0.1. Mu.L was used to level 50. Mu.L of the reaction system.
LFD-RPA detection was performed on pathogens and combinations of different pathogens using different primer probe compositions, the detection results are shown in FIG. 4. As shown in FIG. 4, the result of RPA-LFDs shows that the RPA reaction only takes 10 minutes to obtain the detection result, and the specificity is good.
Example 3 RPA detection of clinical samples
Genomic DNA was extracted from 19 human clinical blood using a blood genomic DNA extraction kit (DP 304, available from Tiangen Biochemical technologies (Beijing) Co., ltd.) and numbered S1-S19. S1-S19 samples were tested by the triple RPA reaction and LFD-RPA reaction of example 2, and the test results are shown in FIG. 5A. The PCR detection is carried out by using bscp-PCR method in national standard and primers bscp-F and bscp-R as reference, and the detection result is shown in FIG. 5B. From the results of A-B in FIG. 5, the blind detection results of the clinical samples detected by the RPA triple fluorescence method and LFDs method are consistent with the detection results of PCR, which shows that the multiple RPA detection and the LFD-RPA detection provided by the invention have good specificity.
Bacillus anthracis is separated from skin exudates of anthrax patients in northern China and named Xa01. Genomic DNA samples of Xa01 strain, A16PI2 strain and A16Q1 strain shown in Table 1 were examined by triple RPA reaction and LFD-RPA reaction in example 2, and the examination results are shown in FIG. 5C. PCR was performed on genomic DNA samples of Xa01 strain, A16PI2 strain and HN001 strain using primers pag-F and pag-R, and on genomic DNA samples of Xa01 strain, A16Q1 strain and HN001 strain using pag and cap using national standard animal anthrax diagnosis technology (NY/T561-2015), and the detection results are shown in FIG. 5D. According to the C-D in FIG. 5, the multiple RPA detection and LFD-RPA detection provided by the invention are consistent with the PCR detection results.
Since a plague clinical sample is difficult to obtain, a certain amount of plague genomic DNA is added to a negative blood genomic sample, the blood genomic sample is simulated, and the blood genomic sample is set to a concentration gradient of 10 5 copies/- μl to 10 0 copies/- μl, with a genomic DNA sample of yersinia enterocolitica 52212 (Yersinia enterocolitica) as a control. The primer probe composition provided by the invention is used for carrying out RPA detection and LFD-RPA detection on blood genome samples with different concentrations, and the detection results are shown as E in figure 5. PCR detection is used as a control, a plague bacillus target spot and RPA detection are used as the same target spot, and PCR detection is carried out on blood genome samples with different concentrations by referring to a plague bacillus and Hantavirus rapid detection method (SN/T2616-2010) carried by a national port mouse, and the plague gene target spot, primers plague-F and plague-R, and the detection results are shown as F in figure 5. According to E-F shown in FIG. 5, the RPA detection, the LFD-RPA detection and the PCR detection are consistent in result, and the same sensitivity as the PCR detection can be achieved, but the required time is less than 1/5 of the PCR time.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
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