CN116716422A - Primer, probe, kit and method for detecting Klebsiella pneumoniae K1 gene and K2 gene - Google Patents
Primer, probe, kit and method for detecting Klebsiella pneumoniae K1 gene and K2 gene Download PDFInfo
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Abstract
The invention relates to the technical field of bacterial detection, and discloses a primer and a probe for detecting Klebsiella pneumoniae K1 genes and K2 genes, wherein the primer is used for amplifying one or two of Klebsiella pneumoniae capsular serotype K1 genes and K2 genes with high toxicity; the probe is used for detecting one or two of a K1 gene and a K2 gene of a high-virulence klebsiella pneumoniae capsular serotype; the primers for amplifying the K1 gene and the K2 gene of the capsule serotypes of the klebsiella pneumoniae with high virulence comprise a forward primer and a reverse primer; the kit for detecting the high-virulence klebsiella pneumoniae capsular serotype K1 genes and the K2 genes by real-time MIRA is suitable for on-site rapid screening of the high-virulence klebsiella pneumoniae capsular serotype K1 genes and the K2 genes; the primer probe for detecting the high-virulence klebsiella pneumoniae capsular serotype K1 genes and the K2 genes by real-time MIRA and the detection method based on the kit can realize rapid and accurate identification of the high-virulence klebsiella pneumoniae capsular serotype K1 genes and the K2 genes in a sample.
Description
Technical Field
The invention relates to the technical field of bacterial detection, in particular to primers, probes, kits and methods for detecting Klebsiella pneumoniae K1 genes and K2 genes.
Background
Klebsiella pneumoniae (Klebsiella pneumoniae, kpn) is a conditional pathogenic bacterium, and when the immunity of the organism is reduced, the infection of respiratory system, urinary system, blood system, soft tissue and the like can be caused frequently; in the middle 80 s of the 20 th century, a unique clinical syndrome of community-acquired, tissue-invasive klebsiella pneumoniae infection, mainly manifested as suppurative liver abscess, with infection foci occurring in multiple sites and possible metastatic spread, was first reported in taiwan region of our country; this newly discovered bacterium is designated as high virulence klebsiella pneumoniae (hypervirulent Klebsiella pneumoniae, hvKp) which can cause serious, potentially fatal infections in relatively healthy hosts, whereas classical klebsiella pneumoniae (classic Klebsiella pneumoniae, cKp) tends to infect immunocompromised populations; hvKp can cause primary infections such as meningitis, necrotizing fasciitis, endophthalmitis, and osteomyelitis, and has a greater infectivity than cKp; in addition, infection by hvKp also has a tendency to metastasize, which is mostly related to the K1 and K2 capsular serotypes and high mucus phenotypes.
At present, the detection methods for the Klebsiella pneumoniae capsular serotype K1 genes and K2 genes mainly comprise a traditional PCR method, a real-time PCR method and the like, and have the defects of long detection period, high technical requirements, complex process and the like; along with the development of molecular biology technology, the establishment of a detection method with rapid response, simple and convenient operation and high sensitivity has important significance for effectively controlling the high-virulence klebsiella pneumoniae.
Disclosure of Invention
The invention aims to provide a primer, a probe, a kit and a method for detecting Klebsiella pneumoniae K1 gene and K2 gene, so as to solve the problems in the background technology.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the invention provides a primer and a probe for detecting Klebsiella pneumoniae K1 genes and K2 genes, wherein the primer is used for amplifying one or two of Klebsiella pneumoniae capsular serotype K1 genes and K2 genes with high toxicity; the probe is used for detecting one or two of a K1 gene and a K2 gene of a high-virulence klebsiella pneumoniae capsular serotype;
the primers for amplifying the K1 gene and the K2 gene of the capsule serotype of the Klebsiella pneumoniae with high virulence comprise a forward primer and a reverse primer, wherein the forward primer is one of K1-F1, K1-F2 and K1-F3 and one of K2-F1, K2-F2 and K2-F3; the reverse primer is one of K1-R1, K1-R2 and K1-R3 and one of K2-R1, K2-R2 and K2-R3;
wherein the nucleotide sequence of K1-F1 is GCTCGCTTGGACGTGCAACAGAAAGATATC;
the nucleotide sequence of K1-F2 is GTCTCTACATGCAGGCATGATACTATGCTCTC;
the nucleotide sequence of K1-F3 is GCATTGTCACAATTTACACGCTTTTTAGAAATAGCC;
the nucleotide sequence of K2-F1 is CGAGCTGGCGCCGCCTTAATTATTATTGTTG;
the nucleotide sequence of K2-F2 is GCTCAATTATTTAGATCCTGCCATAATAAATAAC;
the nucleotide sequence of K2-F3 is GTATTATACAATATAACGATTTTATTAGCCCTG;
the nucleotide sequence of K1-R1 is ACGAGATGTATTTCGTATAACAGTAACAAAAGC;
the nucleotide sequence of K1-R2 is ATAATGAGGAGAACATTACCATAAAACGAGATG;
the nucleotide sequence of K1-R3 is CGTAAAATGATGATATAATAACTGAACTACCC;
the nucleotide sequence of K2-R1 is CAGCAAAACCCTAAACATTAACTTTCCTTTAAGG;
the nucleotide sequence of K2-R2 is GATATTACTATTGAGCATAGCGACAGCAAAAC;
the nucleotide sequence of K2-R3 is CATAGCGACAGCAAAACCCTAAACATTAAC;
the nucleotide sequence of the probe for detecting the high virulence klebsiella pneumoniae capsular serotype K1 gene is as follows: GCATCTATTGCTTATTTACTCGCGAGATTAATAGTTA [ FAM-dT ] AA [ THF ] [ BHQ-dT ] ATTATGATAAGCCTT [ C3spacer ];
the nucleotide sequence of the probe for detecting the high virulence klebsiella pneumoniae capsular serotype K2 gene is as follows: CTACCAGATTGTATGTAGTATCCTTCTATTAGCCG [ ROX-dT ] A [ THF ] [ BHQ-dT ] GTTAAACTGGAGTAC [ C3sp acr ].
As still further aspects of the invention: the forward and reverse primers in the primer are both oligonucleotide sequences.
As still further aspects of the invention: the probe marks dSpacer at a sequence position about 30nt from the 5' end, marks a fluorescent group on a THF site, marks a quenching group on the downstream, the two groups are 2-4nt away, THF is more than or equal to 15nt away from the 3' end, and marks a modification group C3Spacer at the 3' end; the fluorescent groups include FAM and ROX, and the quenching groups include BHQ1 and BHQ2.
As still further aspects of the invention: the nucleotide sequence of the forward primer is
GCTCGCTTGGACGTGCAACAGAAAGATATC and CGAGCTGGCGCCGCCTTAATTATTATTGTTG, the nucleotide sequences of the reverse primers are ACGAGATGTATTTCGTATAACAGTAACAAAAGC and
GATATTACTATTGAGCATAGCGACAGCAAAAC; the nucleotide sequence of the probe for detecting the high virulence klebsiella pneumoniae capsular serotype K1 gene is as follows:
GCATCTATTGCTTATTTACTCGCGAGATTAATAGTTA [ FAM-dT ] AA [ THF ] [ BHQ-dT ] ATTATGATAAGCCTT [ C3spacer ], the nucleotide sequence of the probe for detecting the high virulence Klebsiella pneumoniae capsular serotype K2 gene is as follows:
CTACCAGATTGTATGTAGTATCCTTCTATTAGCCG[ROX-dT]A[THF][BHQ-dT]GTTAAACTGGAGTAC[C3spacer]。
the invention also provides a kit for detecting the Klebsiella pneumoniae K1K2 gene, which comprises a K1 gene, a K2 gene primer, a probe, a dissolving buffer solution, freeze-dried enzyme powder and a magnesium acetate solution.
As still further aspects of the invention: comprises a dissolving buffer solution, freeze-dried enzyme powder, a magnesium acetate solution, a primer and a probe; the dissolving buffer solution comprises 30-50 mM Tris buffer solution and 50-150 mM 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, 100-500 ng/. Mu.L of exonuclease, 50-100 ng/. Mu.L of reverse transcriptase, 1-3 mM ATP, 30-100 mM creatine phosphate, 200-300 ng/. Mu.L of creatine kinase, 200-500 mu.M dNTPs, 5-10% w/v polyethylene glycol 20000 and 1-5 mM dithiothreitol.
The invention also provides a method for detecting Klebsiella pneumoniae K1K2 genes by using the kit, which comprises the following steps:
s1, preparing a reaction system, namely preparing 29.4 mu L of dissolution buffer solution, 1.0 mu L of forward and reverse primers of 10 mu M K gene and K2 gene, 0.3 mu L of probes of 10 mu M K gene and K2 gene, 2.0 mu L of template DNA to be detected, and 11.5 mu L of sterile double distilled water, wherein the total amount of the primers is 47.5 mu L;
s2, adding the prepared reaction system into 50mg of freeze-dried enzyme powder, uniformly mixing, adding 2.5 mu L of 28mM magnesium acetate solution into a tube cover, centrifuging, and reacting at the constant temperature of 39 ℃ for 20min to perform real-time MIRA amplification; observing the detection result by adopting a fluorescence PCR instrument, wherein the K1 gene is a FAM channel, the K2 gene is a ROX channel, and if the FAM channel and the ROX channel both have fluorescence amplification curves, the K1 gene and the K2 gene of the high-virulence Klebsiella pneumoniae capsular serotype exist in the sample to be detected; if only FAM has a fluorescence amplification curve, the existence of a high-virulence Klebsiella pneumoniae capsular serotype K1 gene in the sample to be detected is indicated; if only the ROX channel has a fluorescence amplification curve, the existence of a high-virulence klebsiella pneumoniae capsular serotype K2 gene in the sample to be detected is indicated; otherwise, the test sample is proved to have no high virulence Klebsiella pneumoniae capsular serotype K1 genes and K2 genes.
The real-time MIRA detection method is short in time consumption, high in sensitivity and strong in specificity, and is suitable for on-site rapid screening of the Klebsiella pneumoniae capsular serotype K1 genes and K2 genes.
Compared with the prior art, the invention has the beneficial effects that:
the kit for detecting the high-virulence klebsiella pneumoniae capsular serotype K1 genes and the K2 genes by real-time MIRA provided by the invention is particularly suitable for on-site rapid screening of the high-virulence klebsiella pneumoniae capsular serotype K1 genes and the K2 genes, can provide diagnosis and treatment basis at the first time, improves the treatment rate of infection, and reduces the morbidity and mortality of severe infection and secondary severe infection.
The primer probe for detecting the high-virulence klebsiella pneumoniae capsular serotype K1 gene and the K2 gene and the detection method based on the kit can realize rapid and accurate identification of the high-virulence klebsiella pneumoniae capsular serotype K1 gene and the K2 gene in a sample, and has the advantages of simplicity in operation, short detection time, high sensitivity, strong specificity and the like, and is particularly suitable for basic level and field detection, and has wide application prospect.
The invention has the advantages of short detection time, strong specificity, good repeatability and good practical value.
Drawings
FIG. 1 is a diagram showing the results of primer screening for detecting K1 gene and K2 gene of Klebsiella pneumoniae, primer screening for detecting K1 gene and K2 gene real-time MIRA in the primer screening for detecting K2 gene, probe screening for detecting K2 gene, kit screening for detecting K1 gene and kit screening for detecting K2 gene;
FIG. 2 is a diagram showing the results of the test of the specificity of the K1 gene and the K2 gene reaction in the primers, probes, kits and methods for detecting the K1 gene and the K2 gene of Klebsiella pneumoniae;
FIG. 3 is a diagram showing the results of the sensitivity test of the K1 gene and the K2 gene in the primers, probes, kits and methods for detecting the K1 gene and the K2 gene of Klebsiella pneumoniae;
FIG. 4 is a peak diagram showing the results of electrophoresis and sequencing of K1 gene and K2 gene in primers, probes, kit and method for detecting K1 gene and K2 gene of Klebsiella pneumoniae.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The embodiment of the invention is as follows:
real-time MIRA detection of high virulence Klebsiella pneumoniae capsular serotype K1 gene and K2 gene specific primers, design and screening of probes:
(1) Design of high virulence klebsiella pneumoniae capsular serotype K1 gene and K2 gene specific primers and probes
According to literature reports on nucleic acid sequences of high-virulence klebsiella pneumoniae capsular serotype K1 genes and K2 genes, sequence comparison is carried out on the high-virulence klebsiella pneumoniae capsular serotype K1 genes and the K2 genes, a plurality of corresponding specific primers and probes are designed in a conservation area, and candidate primer and probe sequences are shown in table 1.
Real-time MIRA technical primer design requirement: the primer length is 30-35bp, the base arrangement randomness of the primer sequence is high, the GC content is between 30-70%, the amplified fragment is prevented from forming a secondary structure to influence the amplification, and the length of the amplified fragment is recommended to be 150-300bp, and is usually not more than 500bp; the dSpacer is marked at a sequence position about 30nt from the 5' end, a fluorescent group is marked on the THF site, a quenching group is marked on the downstream, the two groups are 2-4nt away, THF is more than or equal to 15nt from the 3' end, and a modification group C3Spacer is marked on the 3' end.
Candidate primer and probe sequences of Table 1
(2) Screening of high-virulence klebsiella pneumoniae capsular serotype K1 gene and K2 gene specific primers and probes
A. The reaction system: the forward and reverse primers of 29.4. Mu.L, 10. Mu. M K1 gene and K2 gene in the lysis buffer (40 mM Tris buffer, 100mM potassium acetate) were each 1.0. Mu.L, and the probes of 10. Mu. M K1 gene and K2 gene were each 0.3. Mu.L, and 2. Mu.L of the DNA nucleic acid sample of the K1 gene or K2 gene of the capsule serotype of Klebsiella pneumoniae extracted was supplemented with no enzyme water to 50. Mu.L.
B. The prepared reaction system is added into freeze-dried enzyme powder (500 ng/. Mu.L of recombinase, 300 ng/. Mu.L of recombinase cofactor, 400 ng/. Mu.L of single-stranded DNA binding protein, 100 ng/. Mu.L of DNA polymerase, 100 ng/. Mu.L of reverse transcriptase, 3mM ATP, 50mM creatine phosphate, 300 ng/. Mu.L of creatine kinase, 500 mu.M dNTPs, 5.5% w/v polyethylene glycol 20000 and 2mM dithiothreitol), fully dissolved and uniformly mixed, 2.5 mu.L of 28mM magnesium acetate is added into a tube cover, and the mixture is transferred to a constant-temperature amplification integrated detection device for amplification after centrifugation, and the mixture is reacted for 20 minutes at 39 ℃.
C. The detection result is observed by adopting a fluorescence PCR instrument, under the same reaction condition, different primer combinations are amplified effectively, but the amplification effect is different, two indexes of the reaction threshold time and the relative fluorescence intensity are combined, a primer group with an early positive threshold time and a high relative fluorescence unit and a typical S-shaped curve is taken as an optimal primer group, so that the K1 gene optimal primer group is K1-R1, K1-F1 and the K2 gene optimal primer group is K2-R2 and K2-F1, and the primer group is shown in fig. 1 and table 2.
TABLE 2 optimal primers and probes for Real-time MIRA method
(3) Real-time MIRA detection of high virulence Klebsiella pneumoniae capsular serotype K1 Gene and K2 Gene response specificity test
And detecting other pathogens by using the screened optimal primer group and probes, wherein the pathogens comprise Klebsiella pneumoniae, classical Klebsiella pneumoniae, klebsiella oxytoca, escherichia coli, acinetobacter baumannii, pseudomonas aeruginosa and Pseudomonas maltophilia which do not contain K1 genes and K2 genes.
The real-time MIRA detection and the reaction conditions were performed according to the above reaction system, and the results of the specific detection are shown in FIG. 1. Other bacteria are not amplified except the obvious amplification curves of the K1 gene and the K2 gene nucleic acid, and the method can specifically detect the detection virulence Klebsiella pneumoniae capsular serotype K1 gene and the K2 gene and other pathogens without cross reaction, as shown in figure 2.
(4) real-time MIRA detection of high virulence Klebsiella pneumoniae capsular serotype K1 Gene and K2 Gene response sensitivity test
Firstly, 1X 103 CFU-1X 108CFU containing pathogenic bacteria DNA is prepared, real-time MIRA amplification is carried out by taking the pathogenic bacteria DNA as templates, and finally, a fluorescent PCR instrument is adopted to observe the detection result. The results show that the detection limit of the detection method of the invention on the high virulence klebsiella pneumoniae capsular serotype K1 gene and the K2 gene is 1X 103CFU, and the sensitivity of the real-time MIRA is approximately the same as that of the real-time PCR detection method. The sensitivity test results are shown in FIG. 3.
(5) Verifying practicability of real-time MIRA detection method
In order to detect the clinical practicability of the high-virulence klebsiella pneumoniae capsular serotype K1 genes and K2 genes by using the real-time MIRA method kit, 33 strain samples clinically separated in hospitals are detected by using the established real-time MIRA method, and the detection results of all samples are found to be 100% in accordance with the real-time PCR detection results, as shown in table 3, the double PCR method established by the invention is suitable for the rapid detection of the high-virulence klebsiella pneumoniae capsular serotype K1 genes and K2 genes. All high virulence klebsiella pneumoniae capsular serotypes K1 and K2 genes in this experiment were verified by conventional PCR sequencing, and the results of conventional PCR electrophoresis and sequencing are shown in fig. 4.
TABLE 3 clinical validation compliance test results for the inventive kits
hvKp K1: hypervirulent Klebsiella pneumoniae coharboring K1 gene (Klebsiella pneumoniae with high virulence carrying K1 gene);
hvKp K2: hypervirulent Klebsiella pneumoniae coharboring K2 gene (Klebsiella pneumoniae with high virulence carrying K2 gene);
hvKp: hypervirulent Klebsiella pneumoniae non-K1 and non-K2 isopropyl (Klebsiella pneumoniae non-K1 and non-K2 sub-strains);
cKp: classical Klebsiella pneumoniae (classical klebsiella pneumoniae);
ICU: intensive Care Unit (intensive care unit);
GICU: gastroenterology Intensive Care Unit (gastroenterology intensive care unit);
RICU Respiratory Intensive Care Unit (respiratory intensive care unit).
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (9)
1. The primers and the probes for detecting the Klebsiella pneumoniae K1 gene and the K2 gene are characterized in that the primers are used for amplifying one or two of the Klebsiella pneumoniae capsular serotype K1 gene and the K2 gene; the probe is used for detecting one or two of a K1 gene and a K2 gene of a high-virulence klebsiella pneumoniae capsular serotype;
the primers for amplifying the K1 gene and the K2 gene of the capsule serotype of the Klebsiella pneumoniae with high virulence comprise a forward primer and a reverse primer, wherein the forward primer is one of K1-F1, K1-F2 and K1-F3 and one of K2-F1, K2-F2 and K2-F3; the reverse primer is one of K1-R1, K1-R2 and K1-R3 and one of K2-R1, K2-R2 and K2-R3;
wherein the nucleotide sequence of K1-F1 is GCTCGCTTGGACGTGCAACAGAAAGATATC;
the nucleotide sequence of K1-F2 is GTCTCTACATGCAGGCATGATACTATGCTCTC;
the nucleotide sequence of K1-F3 is GCATTGTCACAATTTACACGCTTTTTAGAAATAGCC;
the nucleotide sequence of K2-F1 is CGAGCTGGCGCCGCCTTAATTATTATTGTTG;
the nucleotide sequence of K2-F2 is GCTCAATTATTTAGATCCTGCCATAATAAATAAC;
the nucleotide sequence of K2-F3 is GTATTATACAATATAACGATTTTATTAGCCCTG;
the nucleotide sequence of K1-R1 is ACGAGATGTATTTCGTATAACAGTAACAAAAGC;
the nucleotide sequence of K1-R2 is ATAATGAGGAGAACATTACCATAAAACGAGATG;
the nucleotide sequence of K1-R3 is CGTAAAATGATGATATAATAACTGAACTACCC;
the nucleotide sequence of K2-R1 is CAGCAAAACCCTAAACATTAACTTTCCTTTAAGG;
the nucleotide sequence of K2-R2 is GATATTACTATTGAGCATAGCGACAGCAAAAC;
the nucleotide sequence of K2-R3 is CATAGCGACAGCAAAACCCTAAACATTAAC;
the nucleotide sequence of the probe for detecting the high virulence klebsiella pneumoniae capsular serotype K1 gene is as follows:
GCATCTATTGCTTATTTACTCGCGAGATTAATAGTTA[FAM-dT]AA[THF][BHQ-dT]ATTATGATAAGCCTT[C3spacer]；
the nucleotide sequence of the probe for detecting the high virulence klebsiella pneumoniae capsular serotype K2 gene is as follows:
CTACCAGATTGTATGTAGTATCCTTCTATTAGCCG[ROX-dT]A[THF][BHQ-dT]GTTAAACTGGAGTAC[C3sp acer]。
2. the primers and probes for detecting K1 gene and K2 gene of Klebsiella pneumoniae according to claim 1, wherein the forward and reverse primers in the primers are oligonucleotide sequences.
3. The primer and probe for detecting klebsiella pneumoniae K1 gene and K2 gene according to claim 1, wherein the probe marks dSpacer at a sequence position of about 30nt from the 5' end, marks a fluorescent group on THF site, marks a quenching group on downstream side, the two groups are 2-4nt away, THF is more than or equal to 15nt away from the 3' end, and the 3' end marks a modifying group C3Spacer; the fluorescent groups include FAM and ROX, and the quenching groups include BHQ1 and BHQ2.
4. The primer and probe for detecting Klebsiella pneumoniae K1 gene and K2 gene according to claim 1, wherein the nucleotide sequence of the forward primer is GCTCGCTTGGACGTGCAACAGAAAGATATC and the nucleotide sequence of the reverse primer is ACGAGATGTATTTCGTATAACAGTAACAAAAGC; the nucleotide sequence of the probe for detecting the high virulence klebsiella pneumoniae capsular serotype K1 gene is as follows:
GCATCTATTGCTTATTTACTCGCGAGATTAATAGTTA[FAM-dT]AA[THF][BHQ-dT]ATTATGATAAGCCTT[C3spacer]。
5. the primer and probe for detecting Klebsiella pneumoniae K1 gene and K2 gene according to claim 1, wherein the nucleotide sequence of the forward primer is CGAGCTGGCGCCGCCTTAATTATTATTGTTG and the nucleotide sequence of the reverse primer is GATATTACTATTGAGCATAGCGACAGCAAAAC; the nucleotide sequence of the probe for detecting the high virulence klebsiella pneumoniae capsular serotype K2 gene is as follows:
CTACCAGATTGTATGTAGTATCCTTCTATTAGCCG[ROX-dT]A[THF][BHQ-dT]GTTAAACTGGAGTAC[C3sp acer]。
6. a kit for detecting klebsiella pneumoniae K gene and 1K2 gene, which is characterized by comprising the K1 gene and K2 gene primer and probe according to any one of claims 1-5, and further comprising a dissolution buffer solution, freeze-dried enzyme powder and magnesium acetate solution.
7. The kit for detecting Klebsiella pneumoniae K1 gene and K2 gene according to claim 6, comprising a lysis buffer, lyophilized enzyme powder, magnesium acetate solution, the primer and probe according to any one of claims 2 to 6; the dissolving buffer solution comprises 30-50 mM Tris buffer solution and 50-150 mM 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, 100-500 ng/. Mu.L of exonuclease, 50-100 ng/. Mu.L of reverse transcriptase, 1-3 mM ATP, 30-100 mM creatine phosphate, 200-300 ng/. Mu.L of creatine kinase, 200-500 mu.M dNTPs, 5-10% w/v polyethylene glycol 20000 and 1-5 mM dithiothreitol.
8. A method for detecting klebsiella pneumoniae K1 gene and K2 gene, characterized in that the kit of any one of claims 6 to 7 is used, comprising the steps of:
s1, preparing a reaction system, namely preparing 29.4 mu L of dissolution buffer solution, 1.0 mu L of forward and reverse primers of 10 mu M K gene and K2 gene, 0.3 mu L of probes of 10 mu M K gene and K2 gene, 2.0 mu L of template DNA to be detected, and 11.5 mu L of sterile double distilled water, wherein the total amount of the primers is 47.5 mu L;
s2, adding the prepared reaction system into 50mg of freeze-dried enzyme powder, uniformly mixing, adding 2.5 mu L of 28mM magnesium acetate solution into a tube cover, centrifuging, and carrying out real-time MIRA amplification reaction at constant temperature.
9. The method for detecting K1 and K2 genes of Klebsiella pneumoniae according to claim 8, wherein the reaction temperature is 39℃and the reaction time is 20min.
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| CN119242831A (en) * | 2024-11-15 | 2025-01-03 | 江苏省农业科学院 | A primer-probe combination, a kit and its application for real-time fluorescent MIRA detection of highly virulent Klebsiella pneumoniae |
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| CN119242831A (en) * | 2024-11-15 | 2025-01-03 | 江苏省农业科学院 | A primer-probe combination, a kit and its application for real-time fluorescent MIRA detection of highly virulent Klebsiella pneumoniae |
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