CN116479147A - Primer probe and kit for detecting burkholderia meliotis and application of primer probe and kit - Google Patents
Primer probe and kit for detecting burkholderia meliotis and application of primer probe and kit Download PDFInfo
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a primer probe for detecting burkholderia meliotidis, a kit and application thereof, wherein the primer probe is used for amplifying an ORF1ab gene of burkholderia meliotidis, the primer comprises a forward primer and a reverse primer, the forward primer is one of F1, F2, F3 and F4, and the nucleotide sequence is shown as SEQ ID NO. 1-4; the reverse primer is one of R1, R2, R3 and R4, the nucleotide sequence is shown as SEQ ID NO. 5-8, the probe is P1, and the nucleotide sequence is shown as SEQ ID NO. 9. The invention selects the burkholderia-like ORF1ab gene as a specific target thereof, adopts a recombinase polymerase amplification technology in combination with a lateral chromatography test strip detection method to detect the burkholderia-like, and has the advantages of simple operation, simple and portable required equipment, short time consumption, easy interpretation of results and the like.
Description
Technical Field
The invention belongs to the field of in-vitro nucleic acid molecule detection, and particularly relates to a primer probe for detecting burkholderia melitensis, a kit and application thereof.
Background
The meliosis is a tropical infectious disease caused by the infection of human body by meliosis Bokkalii (Burkholderia pseudomallei, B.pseudomuleii), and is mainly popular in areas such as Thailand, north Australia and Hainan of China. The clinical manifestation of the disease is diversified, various clinical symptoms such as lung infection, acute septicemia, organ abscess and the like are easy to appear, the disease progress is rapid, the prognosis is prolonged, the death rate and the recurrence rate are extremely high, and the disease is an underestimated infectious disease clinically. Human cases of infection of the meliotis occur successively in the tropical areas of Hainan, guangdong and Guangxi since the first report of cases of infection of the meliotis in China in 1990. In the genus burkholderia, however, the colony morphology and immunogenicity of the burkholderia pseudomelitensis are very similar to those of other burkholderia, and the differentiation is difficult. Thus, identification of bacteria having similar morphology and phenotype to Burkholderia and other Burkholderia-like bacteria has been a concern for microbial researchers. Although bacterial isolation culture is a "gold standard" for diagnosing melioidosis, the accuracy of identification by traditional biochemical reaction methods after culture is not high, the time taken for identification is long, and the optimal treatment opportunity is easily missed. ELISA method can be used for detecting specific antibody of Bokkera melitensis, but because of higher antibody positive rate of people in epidemic areas, the method has no great diagnostic value in disease epidemic areas, and the corresponding antibody can not be detected in blood in early stage of acute disease course of melitemia infection. In addition, the antigen of the burkholderia melioides, the burkholderia melioides and other pseudomonas bacteria have common cross antigens, so that the ELISA method for detecting the burkholderia melioides also has the defect of low specificity and the like. The molecular biology method mainly detects the burkholderia melioides and other burkholderia berkholderia by analyzing the bacterial 16S rRNA gene sequence, but the method has the defects of complicated operation steps, easy pollution, long time consumption and the like, and needs expensive instruments and equipment and professional technicians to operate. Therefore, a new method capable of rapidly and accurately identifying burkholderia melitensis and other burkholderia is needed to be researched.
Disclosure of Invention
The invention provides a primer probe for detecting burkholderia melioides, a kit and application thereof, which can not only finish diagnosis and differential diagnosis (less than or equal to 10 min) of the burkholderia melioides, but also judge the detection result (less than or equal to 5 min) by naked eyes, and the whole time is less than or equal to 15min. The invention has simple design and reasonable structure, and has good specificity in the subsequent final result. The invention has the advantages of short detection period, strong specificity, high sensitivity, low omission factor, good repeatability and good practical value.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the invention provides a primer probe for detecting burkholderia melioides, which is used for amplifying an ORF1ab gene of burkholderia melioides, and comprises a forward primer and a reverse primer, wherein the forward primer is one of F1, F2, F3 and F4, and the nucleotide sequence is shown as SEQ ID NO. 1-4; the reverse primer is one of R1, R2, R3 and R4, the nucleotide sequence is shown as SEQ ID NO. 5-8, the probe is P1, and the nucleotide sequence is shown as SEQ ID NO. 9.
Further, the reverse primer is conjugated to biotin at the 5 'end, the probe labels the FAM group at the 5' end, the tetrahydrofuran residue site is 30 nucleotides downstream of the 5 'end and a blocking group dSpacer at the 3' end.
Further, the forward primer is: CTTCAATCTGCTCTTTCCGTTGCTGTGG; the reverse primer is as follows: [ biotin ] CAGGACGGTTTCGGACGAATCCGGAGCGG; the probe is as follows:
[FAM]TCGAGCAATCGGCGGATATCCGGAATCTGG[THF]TCACCACCACTTTCCG[C3Spac er]。
the invention also provides a MIRA kit for detecting the burkholderia meliotidis, which comprises the primer probe, a dissolving buffer solution, freeze-dried enzyme powder, a magnesium acetate solution and a buffer solution.
Further, the lysis buffer comprises 30-50mM Tris buffer and 50-150mM potassium acetate; the freeze-dried enzyme powder comprises 100-500 ng/mu L of recombinase, 100-400 ng/mu L of recombinase cofactor, 400-900 ng/mu L of single-stranded DNA binding protein, 50-200 ng/mu L of DNA polymerase, 1-3mM ATP, 30-100mM creatine phosphate, 200-300 ng/mu L of creatine kinase, 200-500 mu M dNTPs, 5-10% w/v polyethylene glycol 20000 and 1-5mM dithiothreitol; the buffer solution is enzyme-free water or Tris buffer solution; the concentration of the magnesium acetate solution was 280mM.
The invention also provides application of the MIRA kit for detecting the burkholderia melioides, and the kit is used for MIRA amplification detection of an open reading frame (ORF 1 ab) of a burkholderia melioides genome.
Further, the specific detection method comprises the following steps:
1) Extracting total DNA of bacteria to be detected;
2) Taking the extracted total DNA as a template, and carrying out MIRA amplification of the bacteria to be detected;
3) Visual detection is carried out on the MIRA amplification result by adopting a lateral flow test strip (LFD);
4) Analyzing the detection result by observing the condition that the lateral flow test strip has a mauve strip through naked eyes;
further, the step 1) adopts a DNA extraction kit to extract the total DNA of the bacteria to be detected.
Further, the MIRA amplification reaction system in the step 2) comprises: 29.5. Mu.L of dissolution buffer, 2.1. Mu.L of 10. Mu.M forward and reverse primers, 0.6. Mu.L of 10. Mu.M probe, 2.0. Mu.L of template DNA to be detected, 11.2. Mu.L of sterile double distilled water, 50mg of MIRA freeze-dried enzyme powder, 2.5. Mu.L of 280mM magnesium acetate.
Further, the step 2) takes the extracted total DNA as a template, adds the primer, the probe, the dissolving buffer solution and the sterile double distilled water, fully mixes the mixture, adds the mixture into the freeze-dried enzyme powder, then adds the magnesium acetate solution, inverts the mixture, adds the mixture into an EP tube for reaction, places the EP tube into a constant temperature heating module, and the amplification temperature is 34-44 ℃, the amplification time is 6-16min, preferably the amplification temperature is 40 ℃, and the amplification time is 10min.
Further, the step 3) specifically includes: after the reaction of step 2), 10. Mu.L of the amplified product was sucked up and mixed with 190. Mu.L of buffer solution uniformly, and added into a sealed detection tank of a hybrid detect nucleic acid colloidal gold test strip, and the result was observed after standing at room temperature for 5min.
Further, the analysis and detection result of the step 4) by observing the condition that the purplish red strip appears on the lateral flow test strip through naked eyes is specifically as follows: the test strip has two mauve strips, one strip is positioned in the quality control area, and the other strip is positioned in the detection area, and the result is positive, so that the sample contains pathogenic bacteria to be detected; when the test strip has only a mauve strip in the quality control area and no strip exists in the detection area, the result is negative, which indicates that the sample does not contain pathogenic bacteria to be detected.
The invention has the beneficial effects that:
(1) The primer and probe set designed by the invention is used for carrying out MIRA isothermal amplification detection on the open reading frame (ORF 1 ab) of the burkholderia-like genome, and the primer and the probe are designed according to the ORF1ab gene (548 bp) of the burkholderia-like genome. The primer of the invention is used for MIRA-LFD detection, can rapidly, conveniently, efficiently and highly specifically detect the Burkholderia pseudogangrene under isothermal conditions with high sensitivity, does not need complex instruments, and is suitable for rapid screening and detection of the Burkholderia pseudogangrene under basic medical and health units and field environments.
(2) The invention can not only finish the diagnosis and differential diagnosis (less than or equal to 10 min) of the burkholderia meliotidis, but also judge the detection result (less than or equal to 5 min) by naked eyes, and the whole time is less than or equal to 15min. The invention has simple design and reasonable structure, and has good specificity in the subsequent final result. The invention has the advantages of short detection period, strong specificity, high sensitivity, low omission factor, good repeatability and good practical value.
Drawings
FIG. 1 shows 16 pairs of MIRA primer screening results;
FIG. 2 shows the results of different temperature conditions of MIRA-LFD;
FIG. 3 is a graph of the results of optimizing different time conditions of MIRA-LFD;
FIG. 4 shows the sensitivity test results of MIRA-LFD;
FIG. 5 shows the result of specific detection of MIRA-LFD.
Detailed Description
Hereinafter, embodiments of the present invention will be described in detail. The invention can be more clearly understood through the embodiment, and certain changes and modifications can be made on the basis of the invention, so as to obtain different research effects. The reagents involved in the experimental process are all conventional reagents, and the use of the reagents refers to the use instruction of the product.
Examples
(1) Primer probe design, screening and identification of burkholderia-like gangrene
According to the complete gene sequence of the burkholderia-like international standard strain K96243 (ATCC 23344), the ORF1ab gene (548 bp) of the strain is selected for designing primers and probes. According to the invention, 16 pairs of MIRA primers (Table 1) F1/R1, F1/R2, F1/R3, F1/R4, F1/R1, F2/R2, F2/R3, F2/R4, F3/R1, F3/R2, F3/R3, F3/R4, F4/R1, F4/R2, F4/R3, F4/R4 were designed in the ORF1ab gene (GenBank accession number AF 074878), and then the primer pairs were evaluated using the MIRA kit to select the primer set with the highest amplification efficiency and the most single amplification product.
The specific implementation method comprises the following steps:
the kit is used for carrying out MIRA reaction, and the MIRA reaction system comprises: 29.5. Mu.L of lysis buffer, 2.1. Mu.L of forward primer and 2.1. Mu.L of reverse primer (primer concentration 10. Mu.M), 10. Mu.M probe 0.6. Mu.L, 11.2. Mu.L of sterile double distilled water, 2. Mu.L of Burkholderia-like nucleic acid template and 50mg of MIRA lyophilized enzyme powder, 2.5. Mu.L of magnesium acetate (280 mM).
Adding primers, probes, a dissolving buffer solution and sterile double distilled water into a berkovic nucleic acid template of the meliotosus, fully and uniformly mixing, adding into freeze-dried enzyme powder, and finally adding magnesium acetate into a reaction tube to start amplification reaction; after mixing, the reaction solution is thrown (or rapidly centrifuged) to the bottom of the tube, and then the reaction tube is immediately placed into constant temperature equipment for incubation for 10min at 40 ℃; after the completion of the reaction, 50. Mu.L of phenol/chloroform was added thereto, the reaction mixture was extracted at 1:1, centrifuged at 12000rpm for 5 minutes, and 5. Mu.L of the supernatant was subjected to electrophoresis.
The method comprises the steps of selecting a Bruetum melitensis domestic standard strain BPC006 as a template, and screening a MIRA primer by adopting a MIRA basic electrophoresis method, wherein F1/R1, F1/R2, F1/R3, F1/R4, F1/R1, F2/R2, F2/R3, F2/R4, F3/R1, F3/R2, F3/R3, F3/R4, F4/R1, F4/R2, F4/R3 and F4/R4 are shown in lanes 1-16 respectively. The primer combination F1/R1 with better electrophoresis result band is preferably selected for subsequent MIRA-LFD detection. Finally, LFD-F1/R1 and LFD-P1 primer/probe combination are selected for establishing a MIRA-LFD reaction system.
The MIRA-LFD method requires 2 primers and a FAM-labeled probe, wherein the forward primer is conjugated with biotin at the 5 'end, the probe is 46bp in length, the FAM group is labeled at the 5' end, the tetrahydrofuran residue site is 30 nucleotides downstream of the 5 'end and a blocking group (dSpacer) is labeled at the 3' end. 1 reverse primer and 1 probe were redesigned according to the above requirements, the sequences being as follows:
LFD-R1:[biotin]CAGGACGGTTTCGGACGAATCCGGAGCGG；
LFD-P1:[FAM]TCGAGCAATCGGCGGATATCCGGAATCTGG[THF]TCACCACCACTTT CCG[C3Spacer]；
the specific implementation method comprises the following steps:
the MIRA-LFD reaction is carried out by using a MIRA kit, and the MIRA-LFD reaction system comprises: 29.5. Mu.L of lysis buffer, 2.1. Mu.L of upstream primer and 2.1. Mu.L of downstream primer (primer concentration 10. Mu.M), 0.6. Mu.L of probe (probe concentration 10. Mu.M), 11.2. Mu.L of sterile double distilled water, 2. Mu.L of Burkholderia meliotidis nucleic acid template and 50mg of MIRA lyophilized enzyme powder, and finally 2.5. Mu.L of magnesium acetate (280 mM) is added to the reaction tube to initiate the amplification reaction.
Adding primers, probes, a dissolving buffer solution and sterile double distilled water into a berkovic nucleic acid template of the meliotosus, fully and uniformly mixing, adding into freeze-dried enzyme powder, and finally adding magnesium acetate into a reaction tube to start amplification reaction; after uniform mixing, the reaction solution is thrown (or rapidly centrifuged) to the bottom of the tube, and then the reaction tube is immediately placed into constant temperature equipment for incubation; after the reaction, 10. Mu.L of the amplification product and 190. Mu.L of buffer were mixed uniformly and added to a sealed hybrid detect nucleic acid colloidal gold test strip detection tank, and the experimental result was read.
Incubation temperature and time screening: firstly, respectively incubating a reaction system at 40 ℃ for 6, 8, 10, 12, 14 and 16min, stopping the reaction by placing a reaction tube on ice at corresponding time, diluting a product in PBS, reading a result as described above, and further judging the optimal reaction time; then, the amplification reaction was carried out at 34℃at 36℃at 38℃at 40℃at 42℃at 44℃for 10 minutes, and the optimum reaction temperature was determined.
By screening the detection temperature and time, no difference in detection effect between 34-44 ℃ and 6-16min is obtained, see fig. 2 and 3, and finally 40 ℃ and 10min are preferable as reaction conditions.
(2) Sensitivity test
The Brucella melitensis-like domestic standard strain BPC006 is used as positive quality control, and the negative control is H 2 O. To assess the sensitivity of the MIRA-LFD detection method, working standards of different concentration gradients were used as templates and incubated for 10min at 40 ℃. LOD represents the lowest detectable amount of DNA that shows a clear band on the test strip. For comparison, diluted DNA samples were tested in parallel by the TaqMan fluorescent quantitative PCR method reported in the previous study.
The sensitivity of the MIRA-LFD method for detecting the Burkholderia-like bacteria is found to be 3 pg/mu L, as shown in figure 4, and the sensitivity is higher than that of the traditional research.
(3) Specificity test
To verify the specificity of the MIRA-LFD detection method, the DNA (the concentration range of the strain DNA is 0.98-2.69 ng/strain/reaction) of the Burkholderia melitensis, burkholderia taenii, burkholderia cepacia, stenotrophomonas maltophilia, pseudomonas aeruginosa, acinetobacter baumannii, klebsiella pneumoniae and Escherichia coli is selected as a template for the MIRA-LFD reaction. The above experiments were repeated twice.
The specificity test results are shown in fig. 5, and the test results of 7 non-detection bacteria are negative and do not cross react with the detection bacteria. Finally, the establishment of the MIRA-LFD method is utilized to verify the accuracy of clinical identification, and the capability of the MIRA-LFD method for detecting the burkholderia melitensis is completely consistent with the positive result of PCR identification.
Table 1 primer and probe sequences of Burkholderia meliotidis ORF1ab gene and modification thereof
The lysis buffer in this example comprises 30-50mM Tris buffer and 50-150mM potassium acetate; the freeze-dried enzyme powder comprises 100-500 ng/mu L of recombinase, 100-400 ng/mu L of recombinase cofactor, 400-900 ng/mu L of single-stranded DNA binding protein, 50-200 ng/mu L of DNA polymerase, 1-3mM ATP, 30-100mM creatine phosphate, 200-300 ng/mu L of creatine kinase, 200-500 mu M dNTPs, 5-10% w/v polyethylene glycol 20000 and 1-5mM dithiothreitol; the buffer solution is enzyme-free water or Tris buffer solution. The analysis and detection results of the condition that the purplish red strip appears by observing the lateral flow test strip by naked eyes are specifically as follows: the test strip has two mauve strips, one strip is positioned in the quality control area, and the other strip is positioned in the detection area, and the result is positive, so that the sample contains pathogenic bacteria to be detected; when the test strip has only a mauve strip in the quality control area and no strip exists in the detection area, the result is negative, which indicates that the sample does not contain pathogenic bacteria to be detected.
The technical scheme of the invention is explained in the technical scheme, the protection scope of the invention cannot be limited by the technical scheme, and any changes and modifications to the technical scheme according to the technical substance of the invention belong to the protection scope of the technical scheme of the invention.
Claims (10)
1. The primer probe for detecting the burkholderia melioides is characterized in that: the primer probe is used for amplifying the burkholderia melioides ORF1ab gene, the primer comprises a forward primer and a reverse primer, the forward primer is one of F1, F2, F3 and F4, and the nucleotide sequence is shown as SEQ ID NO. 1-4; the reverse primer is one of R1, R2, R3 and R4, the nucleotide sequence is shown as SEQ ID NO. 5-8, the probe is P1, and the nucleotide sequence is shown as SEQ ID NO. 9.
2. The primer probe for detecting burkholderia melioides according to claim 1, wherein: the reverse primer was conjugated to biotin at the 5 'end, the probe labeled FAM group at the 5' end, the tetrahydrofuran residue site 30 nucleotides downstream of the 5 'end and a blocking group dSpacer at the 3' end.
3. The primer probe for detecting burkholderia melioides according to claim 1, wherein: the forward primer is as follows: CTTCAATCTGCTCTTTCCGTTGCTGTGG; the reverse primer is as follows:
[ biotin ] CAGGACGGTTTCGGACGAATCCGGAGCGG; the probe is as follows:
[FAM]TCGAGCAATCGGCGGATATCCGGAATCTGG[THF]TCACCACCACTTTCCG[C3Spac er]。
4. MIRA kit for detecting burkholderia melioides, which is characterized in that: the MIRA kit comprises the primer probe according to any one of claims 1 to 3, a dissolving buffer, freeze-dried enzyme powder, a magnesium acetate solution and a buffer.
5. The MIRA kit for detecting burkholderia meldonii of claim 4, wherein: the dissolving buffer comprises 30-50mM Tris buffer and 50-150mM potassium acetate; the freeze-dried enzyme powder comprises 100-500 ng/mu L of recombinase, 100-400 ng/mu L of recombinase cofactor, 400-900 ng/mu L of single-stranded DNA binding protein, 50-200 ng/mu L of DNA polymerase, 1-3mM ATP, 30-100mM creatine phosphate, 200-300 ng/mu L of creatine kinase, 200-500 mu M dNTPs, 5-10% w/v polyethylene glycol 20000 and 1-5mM dithiothreitol; the buffer solution is enzyme-free water or Tris buffer solution; the concentration of the magnesium acetate solution was 280mM.
6. The application of the MIRA kit for detecting the burkholderia melioides is characterized in that: the MIRA kit of claim 4 or 5 for MIRA amplification detection of the open reading frame of the burkholderia meliotidis genome (ORF 1 ab).
7. The use of a MIRA kit for detecting burkholderia meldonii according to claim 3, wherein: the specific detection method comprises the following steps:
1) Extracting total DNA of bacteria to be detected;
2) Taking the extracted total DNA as a template, and carrying out MIRA amplification of the bacteria to be detected;
3) Visual detection is carried out on the MIRA amplification result by adopting a lateral flow test strip (LFD);
4) And analyzing the detection result by observing the condition that the purplish red strip appears on the lateral flow test strip by naked eyes.
8. The use of a MIRA kit for detecting burkholderia meldonii according to claim 7, characterized in that: the MIRA amplification reaction system in the step 2) comprises the following steps: 29.5 mu L of dissolution buffer, 2.1 mu L of 10 mu M forward and reverse primers respectively, 0.6 mu L of 10 mu M probe, 2.0 mu L of template DNA to be detected, 11.2 mu L of sterile double distilled water, 50mg of freeze-dried enzyme powder and 2.5 mu L of 280mM magnesium acetate;
and 2) taking the extracted total DNA as a template, adding a primer, a probe, a dissolving buffer solution and sterile double distilled water, fully and uniformly mixing, adding the mixture into freeze-dried enzyme powder, then adding a magnesium acetate solution, uniformly mixing, and performing an amplification reaction at 34-44 ℃ for 6-16min.
9. The use of a MIRA kit for detecting burkholderia meldonii according to claim 7, characterized in that: the step 3) is specifically as follows: after the reaction of step 2), 10. Mu.L of the amplified product was sucked up and mixed with 190. Mu.L of buffer solution uniformly, and added into a sealed detection tank of a hybrid detect nucleic acid colloidal gold test strip, and the result was observed after standing at room temperature for 5min.
10. The use of a MIRA kit for detecting burkholderia meldonii according to claim 7, characterized in that: the analysis and detection result of the condition that the purple red strip appears on the lateral flow test strip through naked eyes is specifically as follows: the test strip has two mauve strips, one strip is positioned in the quality control area, and the other strip is positioned in the detection area, and the result is positive, so that the sample contains pathogenic bacteria to be detected; when the test strip has only a mauve strip in the quality control area and no strip exists in the detection area, the result is negative, which indicates that the sample does not contain pathogenic bacteria to be detected.
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