CN110628923A - Primer combination, kit and PSR method for detecting Klebsiella pneumoniae - Google Patents
Primer combination, kit and PSR method for detecting Klebsiella pneumoniae Download PDFInfo
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Abstract
The invention discloses a primer combination, a kit and a PSR (phosphoenolpyruvate carboxylase) method for detecting Klebsiella pneumoniae, and belongs to the technical field of biological detection. The invention can detect the Klebsiella pneumoniae by a calcein color development method, a pH indicator color development method, a turbidity method or a fluorescence method. The detection method of the invention has simple operation, short reaction time, high sensitivity which is 10 times of that of the common PCR reaction, strong specificity and can only detect the Klebsiella pneumoniae.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a primer combination, a kit and a PSR (phospho-synthesis region) method for detecting Klebsiella pneumoniae.
Background
Klebsiella pneumoniae (Klebsiella pneumoniae) is a major gram-negative bacterium causing in vivo infection, which causes pneumonia, urinary tract and wound infection, and has a high incidence rate in infants, diabetic patients, tumor patients, patients who have long-term antibiotic use, and the elderly. In addition, the increasingly common drug-resistant Klebsiella pneumoniae has become a clinically important problem in recent years, and thus a rapid and sensitive detection method needs to be established.
The traditional detection methods of klebsiella pneumoniae include a microbiological microscopic examination method, a biochemical identification method and a latest full-automatic bacteria identification device based on phenotype and physiological and biochemical indexes, but the methods are long in time consumption and low in sensitivity, and often require several days of bacteria culture time. In recent years, a series of newly invented molecular biological techniques have been used for detecting Klebsiella pneumoniae, such as the detection of Klebsiella pneumoniae in infant milk powder by PCR based on 16S-23S internal transcribed spacer, and there are also triple PCR and real-time fluorescent quantitative PCR techniques for detecting Klebsiella pneumoniae, however, these techniques are relatively complicated, require specialized and expensive instruments, and furthermore Taq enzyme used in PCR is easily inhibited by inhibitors in the original sample and inactivated.
Therefore, it is a problem to be solved by those skilled in the art to provide a method for detecting klebsiella pneumoniae by polymerase helix reaction (PSR).
Disclosure of Invention
In view of the above, the invention provides a primer combination, a kit and a PSR method for detecting Klebsiella pneumoniae, which are simple to operate, high in sensitivity and strong in specificity.
In order to achieve the purpose, the invention adopts the following technical scheme:
capsular Polysaccharide (CPS) is an important pathogenic factor of Klebsiella pneumoniae, rcsA gene regulates the synthesis of capsular polysaccharide and is a specific gene of Klebsiella pneumoniae, so that the invention selects the RCsA gene as a target gene for Klebsiella pneumoniae detection, designs a set of PSR primers according to the rcsA gene sequence, evaluates the optimal temperature, specificity and sensitivity of the PSR primers, and finally applies the method after use to sample detection.
A primer combination for detecting Klebsiella pneumoniae has the following specific primer sequences:
rcsA-Ft1：5’-CGACGTACAGTGTTTCTGCAGTAAAAAACAGGAAATCGTTGAGG-3’；SEQ ID NO.1；
rcsA-Bt1：5’-CGACGTACAGTGTTTCTGCAGGCGAATAATGCCATTACTTTC-3’；SEQ ID NO.2；
rcsA-IF：5’-ATCCGCAGCACTGTTGA-3’；SEQ ID NO.9；
rcsA-IB：5’-GAAGACTGTTTCGTGCATGATGA-3’；SEQ ID NO.10。
further, a kit comprising the above primer combination.
A PSR method for detecting klebsiella pneumoniae, comprising the steps of: and carrying out PSR reaction on the sample to be detected by using the primer combination, detecting a PSR reaction product, and determining whether the sample to be detected is Klebsiella pneumoniae or not.
Further, the PSR reaction system is as follows: mu.l of 2 XMM, 2.0. mu.l of DNA template and 1.0. mu.l of Bst DNA polymerase, 1.6. mu.M each of rcsA-Ft1, rcsA-Bt1, rcsA-IF and rcsA-IB, of primers, and 25. mu.l of water were made up.
Further, the PSR reaction conditions are 65 ℃ and 60 min.
Further, the method for determining whether the sample to be detected is klebsiella pneumoniae is a, b, c or d:
a. calcein color development method
Adding 1.0 mul of calcein indicator into a PSR reaction system; after the isothermal amplification reaction is finished, if the liquid of the reaction tube of the sample to be detected is yellow-green, the detection result of the sample is positive, wherein the detection result indicates that the klebsiella pneumoniae is detected; if the liquid of the reaction tube of the sample to be detected is orange red, the detection result is negative, and the detection result indicates that the Klebsiella pneumoniae is not detected;
b. pH indicator color development method
Add 1.0. mu.l of pH indicator (0.08mM cresol red and 0.02mM phenol red) to the PSR reaction; after the isothermal amplification reaction is finished, if the liquid of the reaction tube of the sample to be detected is yellow, the existence of the klebsiella pneumoniae in the sample to be detected is indicated; if the liquid of the reaction tube of the sample to be detected is red, the existence of Klebsiella pneumoniae in the sample to be detected is indicated; if the negative control tube is changed into yellow or the color of the positive control tube is not changed within 50 minutes, the detection result is invalid, and the detection is required to be carried out again;
c. turbidity method
Detecting a turbidity change curve of a PSR reaction product of the sample to be detected by a turbidity meter, wherein if the turbidity change curve of the PSR reaction product of the sample to be detected is an S-shaped reaction curve, Klebsiella pneumoniae exists in the sample to be detected, and if the turbidity change curve of the PSR reaction product of the sample to be detected does not form an obvious S-shaped reaction curve, the Klebsiella pneumoniae does not exist in the sample to be detected;
d. fluorescence method
The positive reaction appeared as: an S-shaped amplification curve appears in the reaction stage, and the dissolution curve is a single peak; the appearance of negative reactions was: no amplification curve exists in the reaction stage, and no peak appears in the dissolution curve; and (4) result judgment standard: under the premise that the positive control has a positive reaction and the negative control has a negative reaction, the sample to be detected has an S-shaped amplification curve in the reaction stage, the dissolution curve is a single peak and is close to the position of the positive control single peak (the Tm change range is within 1-3 ℃), the sample to be detected is judged to be positive, and otherwise, the sample to be detected is negative; if the positive sample to be detected has negative reaction or the negative sample to be detected has positive reaction, the detection should be carried out again.
Calcein color development method: calcein is a metal chelator; calcein with Mn before reaction2+Complexing of Mn2+Quenching the fluorescence of calcein, making the reaction solution orange red, and making magnesium in the solution in divalent ion state without replacing Mn2+(ii) a When the PSR reaction occurs, a large amount of pyrophosphatase precipitate is formed, and Mn is present2+Will be engaged withMg2P2O7Undergoes a replacement reaction to generate Mn2P2O7And Mg2+And Mg2+Without quenching the calcein fluorescence, the fluorescence is released so that the solution changes from orange to green. When the reaction is negative, the solution keeps the orange red color unchanged; the yellow-green color after the reaction is judged to be positive, and the orange-red color is judged to be negative.
And (3) detecting by a pH indicator color development method: and after the constant-temperature amplification reaction is finished, cooling to room temperature, observing the result, wherein the detection tube turns yellow to indicate that the Klebsiella pneumoniae gene (positive) exists in the sample to be detected, and the color of the detection tube does not change (red) to indicate that the Klebsiella pneumoniae gene (negative) does not exist in the sample to be detected. If the negative control tube is changed into yellow or the color of the positive control tube is not changed (red) within 50 minutes, the detection result is invalid, and the detection is required to be carried out again.
The detection principle of the pH indicator color development method is as follows: when a DNA polymerase binds a deoxyribonucleotide molecule to a newly synthesized DNA double strand, a hydrogen ion is generated as a by-product, and as the PSR reaction proceeds, a large amount of hydrogen ions are generated, and the pH of the reaction system decreases as the concentration of the hydrogen ion increases. When the pH indicator solution was added to 25. mu.l of the PSR reaction system, the positive reaction became yellow, and the negative control solution remained red.
The turbidity method detection principle is as follows: mg formation during the PSR reaction2P2O7I.e. magnesium pyrophosphate, as a white precipitate, the reaction formula occurs as follows:
(DNA)n-1+dNTP→(DNA)n+P2O7 4-
P2O7 4-+2Mg2+-→Mg2 P2O7↓。
according to the reaction principle, the real-time turbidimeter LA-320c measures the turbidity of the reaction tube every 6 seconds and draws a curve to judge whether the reaction is positive or negative; forming a reaction curve-S-shaped broken line, and then the reaction curve is positive; no obvious reaction curve is formed-no S-shaped broken line, the reaction is negative.
The fluorescence real-time detection method comprises the following steps: setting the absorption wavelength to be about 497nm and the maximum emission wavelength to be about 520nm, wherein the wavelength is the same as SYBR Green I and is Green fluorescence; the reaction system was placed in a fluorescence quantitative instrument and the reaction was started.
The positive reaction appeared as: an S-shaped amplification curve appears in the reaction stage, and the dissolution curve is a single peak; the appearance of negative reactions was: there was no amplification curve during the reaction phase and no peak in the dissolution curve. And (4) result judgment standard: under the premise that the positive control has a positive reaction and the negative control has a negative reaction, the sample has an S-shaped amplification curve in the reaction stage, the dissolution curve is a single peak and is close to the positive control single peak (the Tm change range is within 1-3 ℃), the sample can be judged to be positive at this moment, and the sample is negative otherwise. If a negative reaction occurs in the positive sample or a positive reaction occurs in the negative sample, the experiment should be repeated.
The principle of the fluorescence real-time detection method is as follows: the nucleic acid fluorescent dye can be embedded into a DNA double strand, and can emit fluorescence under the excitation of fluorescence with a specific excitation wavelength so as to be detected by an instrument.
According to the technical scheme, compared with the prior art, the primer combination, the kit and the PSR method for detecting the Klebsiella pneumoniae are simple and convenient to operate, short in reaction time, high in sensitivity, 10 times of that of a common PCR reaction, strong in specificity and only capable of detecting the Klebsiella pneumoniae.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a diagram showing PSR optimal primers for screening Klebsiella pneumoniae according to the present invention;
FIG. 2 is a schematic diagram of a specificity-real-time turbidity method for detecting Klebsiella pneumoniae PSR according to the present invention;
FIG. 3 is a schematic diagram of a calcein color development method for detecting specificity of Klebsiella pneumoniae PSR according to the invention;
FIG. 4 is a diagram showing the specificity of detection of Klebsiella pneumoniae PSR-pH indicator color development method of the present invention;
among these, fig. 2-fig. 4, 1, positive control (k. pneumoconiae ATCC BAA-2146); 2, negative control (pure water); 3, Klebsiella oxytoca ATCC 700324; 4, Klebsiella rhizomucosis CMCC 46111; 5, Citrobacterfreundii CMCC 48001; 6, Proteus mirabilis CMCC 49005; 7, Proteus vulgaris CMCC 49027; 8, Serratia marcocens ATCC 14756; 9, morganella amorganii ATCC 25830; 10, Enterobacter aerogenes ATCC 13048; 11, enterobacter cloacae ATCC 13047; 12, Streptococcus pneumoniae 112-07; 13, mycobacteria cubulosis 005; 14, Pseudomonas aeruginosa D104; 15, haempohilus influenza ATCC 49247; 16, Yersinia enterocolitica 027; 17, Yersinia pestis 2638; 18, bacillus specimen 3450; 19, Vibrio cholera 3802; 20, Salmonella aberdeen 9264; 21, neissemia meningitides CMCC 29022; 22, Staphylococcus aureus 2740; 23, Pseudomonas pseudomallei 029; 24, salmonella typhimurium 4030; 25, Corynebacterium diphtheriae CMCC 38001; 26, Bacillus megatherium 4623; 27, Stenotrophomonas maltophilia K279 a; 28, legioniella pneumophila 9135; 29, Acinetobacter baumannii 12101; 30, enteroinvasive e.coli 44825; 31, enterotoxigenic e.coli 44824; 32, enterpathogenic e.coli 2348;
FIG. 5 is a schematic diagram showing the sensitivity detection-turbidity method of the Klebsiella pneumoniae PSR method of the present invention;
FIG. 6 is a calcein color development method for detecting sensitivity of Klebsiella pneumoniae PSR method according to the present invention;
FIG. 7 is a schematic diagram showing the sensitivity detection-pH indicator color development method for the Klebsiella pneumoniae PSR method according to the present invention;
FIG. 8 is a diagram showing the sensitivity detection of Klebsiella pneumoniae by a general PCR method according to the present invention;
wherein, in FIGS. 5-8, 1, negative control (pure water); 2,115.0 ng/l; 3, 11.5 ng/l; 4, 1.15 ng/l; 5,115.0 pg/l; 6, 11.5 pg/l; 7, 1.15 pg/l; 8, 0.115 pg/l; the size of the PCR target fragment is 176 bp;
FIG. 9 is a graph showing fluorescence sensitivity assay for Klebsiella pneumoniae in clinical samples according to the invention;
wherein, 1, 115.0 ng/l; 2, 11.5 ng/l; 3, 1.15 ng/l; 4,115.0 pg/l; 7, 11.5 pg/l; 5, 1.15 pg/l; 6, 0.115 pg/l; 8, negative control (pure water);
FIG. 10 is a graph showing the detection of Klebsiella pneumoniae calcein by the chromogenic method in a clinical sample according to the present invention;
among them, 1, negative control (pure water); 2, positive control (k. pneumoconiae ATCC BAA-2146); 3-34 are different numbers of 32 Klebsiella pneumoniae isolated from suspected infected patients: 3, k.pneumoniae WJ-48; 4, k.pneumoniae WJ-50; 5, k.pneumoniae WJ-51; 6, k.pneumoniae WJ-52; 7, k.pneumoniae wj-53; (ii) 8, k.pneumoniae WJ-57; 9, k.pneumoniae WJ-58; 10, k.pneumoniae WJ-60; 11, k.pneumoniae WJ-61; 12, k.pneumoniae WJ-64; 13, k.pneumoniae WJ-65; 14, k.pneumoniae WJ-66; 15, k.pneumoniae WJ-68; 16, k.pneumoniae 301-; 17, k.pneumoniae 301-; 18, k.pneumoniae 301-; 19, k.pneumoniae 301-; 20, k.pneumoniae 301-; 21, k.pneumoniae 301-; 22, k. pneumoniae 301-; 23, k.pneumoniae 301-; 24, k. pneumoniae 301-; 25, k.pneumoniae 301-; 26, k.pneumoniae 307-; 27, k.pneumoniae 307-; 28, k.pneumoniae 307-429; 29, k.pneumoniae 307-095; 30, k.pneumoniae 307-003; 31, k.pneumoniae 307-; 32, k.pneumoniae 307-; 33, k.pneumoniae 307-; 34, k. pneumoniae 307-.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Preparation of Reaction Mixture (2 XRM)
(1) Preparation of 20 × RM pre: 2.64g of ammonium sulfate, 1.49g of potassium chloride, 3.95g of magnesium sulfate heptahydrate and 2.0ml of Tween 20 are fully dissolved in a 100ml volumetric flask, the pH value is adjusted to 8.0 by using 1% KOH solution, finally the volume is fixed to 100ml, and the mixture is stored at 4 ℃.
(2) Preparation of betaine solution (0.375 g/ml): taking 7.5g betaine in a 20ml volumetric flask, fully dissolving and fixing the volume to 20ml, and storing at 4 ℃.
(3) Preparation of 2 × RM: the reaction buffer solution of PSR should avoid repeated freeze thawing, so it is generally prepared into 2ml small system; mu.l of 20 XMM pre was pipetted into a 2ml centrifuge tube, 53.33. mu.l of Tris-HCl solution (1.5M, pH8.8), 1.0ml of betaine solution (0.375g/ml) and 746.67. mu.l of deionized water were added to make up 2ml, and the tube was stored at-20 ℃.
Bst DNA Large fragment polymerase (NEB Co.); 2 × Taq PCR Mastermix (Beijing Tiangen).
Example 1 PSR reaction
The PSR reaction system adopts a 25-microliter system: comprises 12.5. mu.l of 2 XMM, 2.0. mu.l of DNA template and 1.0. mu.l of Bst DNA polymerase, 1.6. mu.M for Ft and Bt and 0.8. mu.M for IF and IB, and 25. mu.l of water is added.
When the calcein is used for color development judgment, 1.0 mul of calcein indicator is added into a PSR reaction system, and the total volume is 26 mul.
When a pH indicator was used for color discrimination, 1.0. mu.l of a pH indicator (0.08mM cresol red and 0.02mM phenol red) was added in a total volume of 26. mu.l.
The PSR reaction program was 65 ℃ for 60 min.
The PSR reaction analysis is used for detecting the optimal primer, specificity and sensitivity of the Klebsiella pneumoniae.
Example 2 Klebsiella pneumoniae PSR primer design
The sequence of the rcsA gene of the Klebsiella pneumoniae comes from a Genebank website (Accession Number: 7946097) and is uploaded to a Blast website for sequence comparison, and the comparison results show that the rcsA gene is Klebsiella pneumoniae, which shows that the rcsA gene has good specificity to the Klebsiella pneumoniae and can be used for designing a specific PSR primer for detecting the Klebsiella pneumoniae.
4 sets of PSR primers (KP-1, KP-2, KP-3 and KP-4) are designed aiming at the rcsA gene of the Klebsiella pneumoniae, a pair of accelerating primers (IF and IB) are shared, and the names and sequences of the primers are shown in Table 1.
TABLE 1 set of PSR primers designed for rcsA Gene
The reaction was carried out at 65 ℃ for 60min, and the results of comparing the 4 sets of primers in Table 1 are shown in FIG. 1, which shows that the Ct value of the KP-1 primer is the smallest, and thus it was used as the best PSR primer for the detection of Klebsiella pneumoniae.
Example 3 specificity of detection of Klebsiella pneumoniae PSR
To evaluate the specificity of the PSR reaction in detecting Klebsiella pneumoniae, Klebsiella pneumoniae ATCC BAA-2146 and Klebsiella oxytoca ATCC 700324, Klebsiella rhinosclerosus Klebsiella nonscalmatis CMCC46111, Klebsiella Citrobacter freonii Freundii CMCC 48001, Proteus mirabilis CMCC 49005, Proteus vulgaris CMCC49027, Serratia marcescens ATCC 14756, Morganella morganii ATCC 25830, Enterobacter aerogenes ATCC 13048, Enterobacter clobacter cloacae ATCC 13047, Streptococcus pneumoniae Pseudomonas 112-07, Salmonella typhimurium ATCC 34104, Yersinia pestis ATCC 38038, Klebsiella pneumoniae Bacillus subtilis ATCC 380247, Klebsiella pneumoniae Yersinia pestis ATCC 38038, Escherichia coli Yersinia jejunipes ATCC 380247, Escherichia coli Yersinia jejunipes ATCC 34247, Escherichia coli Yersinia pestis ATCC 34104, Yersinia jejunipes, The genome of Diplococcus meningitidis Neisseria meningitidis CMCC 29022, Staphylococcus aureus 2740, Pseudomonas pseudorhinopteroides Pseudomonas pseudomonads 029, Salmonella typhimurium 4030, Corynebacterium diphtheriae diphenoxy CMCC38001, Bacillus megaterium 4623, stenotrophomonas K279a, Legionella pneumophila Legionnella 9135, Acinetobacter baumannii 12101, Escherichia coli invasive E.coli 44825, Escherichia coli pathogenic E.coli 44824, Escherichia coli pathogenic E.coli 448, etc. was extracted by a method and detected by a non-invasive method using a P.pneumoniae PCR primer R2348. The results show that the real-time turbidity method (fig. 2), the calcein color method (fig. 3) and the pH indicator color method (fig. 4) both accurately identify klebsiella pneumoniae, and other bacteria of the same genus as klebsiella or other genera are negative results along with pure water, indicating that the PSR method has good specificity in detecting klebsiella pneumoniae.
Example 4 comparison of PSR with PCR sensitivity
To compare the sensitivity of PSR (including real-time turbidity method and color development method) to conventional PCR method in detecting Klebsiella pneumoniae, K.pneumoconiae ATCC BAA-2146 genomic nucleic acid was diluted with pure water in 10-fold gradient from 115.0 ng/. mu.l to 0.115 pg/. mu.l, and 2.0. mu.l of the template was sampled.
A25. mu.l system was used for PCR reaction, including 12.5. mu.l of PCR MasterMix, 0.5M each of the upstream primer KP-F and the downstream primer KP-B, and the DNA template used was the same as that used for the PSR reaction, and 25. mu.l was filled with pure water. The PCR annealing temperature was set at 55 ℃ and the products were horizontally electrophoresed on 1% agarose gel (Amresco) and photographed with an ultraviolet imager (Bio-Rad).
Wherein, the sequences of the upstream primer and the downstream primer are as follows:
KP-F：5’-GGATATCTGACCAGTCGG-3’；SEQ ID NO.11；
KP-B：5’-GGGTTTTGCGTAATGATCTG-3’；SEQ ID NO.12。
the turbidimetry was monitored every 6s at 650nm wavelength using a real-time turbidimeter, the chromogenic method was performed in a thermostated metal bath, and 1. mu.l calcein indicator was added to 25. mu.l PSR reaction system before the reaction started. The results showed that the fifth gradient, 11.5 pg/. mu.l, was detected by the turbidity method (FIG. 5), the color development method (FIGS. 6-7) and the fluorescence method (FIG. 9) of PSR, while the fifth gradient (lane 6) of the conventional PCR method (FIG. 8) was not evident, although it was a band, indicating that the PSR method was 10 times more sensitive than the conventional PCR in detecting Klebsiella pneumoniae.
Example 5 application of PSR method in clinical detection of Klebsiella pneumoniae
The effect of PSR in clinical detection of Klebsiella pneumoniae was evaluated by using sputum samples of 110 suspected multiply-infected ICU patients and 10 healthy human volunteers, and all samples were analyzed by using the PSR method (calcein color development method) and the ordinary PCR method, the results are shown in FIG. 10, among 110 clinical samples, 32 samples are positive in PSR, 25 samples are positive in PCR, all PCR positive samples are detected by PSR, and all the healthy human sputum samples are negative in PSR method and PCR method. From these 32 PSR positive sputum samples, 32 klebsiella pneumoniae were successfully isolated, and the rcsA gene sequencing results were consistent with Blast website.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
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Claims (6)
1. A primer combination for detecting Klebsiella pneumoniae is characterized in that the specific primer sequence is as follows:
rcsA-Ft1：5’-CGACGTACAGTGTTTCTGCAGTAAAAAACAGGAAAT CGTTGAGG-3’；SEQ ID NO.1；
rcsA-Bt1：5’-CGACGTACAGTGTTTCTGCAGGCGAATAATGCCATTAC TTTC-3’；SEQ ID NO.2；
rcsA-IF：5’-ATCCGCAGCACTGTTGA-3’；SEQ ID NO.9；
rcsA-IB：5’-GAAGACTGTTTCGTGCATGATGA-3’；SEQ ID NO.10。
2. a kit comprising the primer combination of claim 1.
3. A PSR method for detecting klebsiella pneumoniae, comprising the steps of: performing a PSR reaction on a sample to be tested by using the primer combination of claim 1, detecting a PSR reaction product, and determining whether the sample to be tested is Klebsiella pneumoniae.
4. The PSR method for detecting Klebsiella pneumoniae according to claim 3, wherein the PSR reaction system comprises: mu.l of 2 XMM, 2.0. mu.l of DNA template and 1.0. mu.l of BstDNA polymerase, 1.6. mu.M each of rcsA-Ft1, rcsA-Bt1, rcsA-IF and rcsA-IB, of primers, and 25. mu.l of water were made up.
5. The PSR method for detecting Klebsiella pneumoniae according to claim 3, wherein the PSR reaction condition is 65 ℃ and 60 min.
6. The PSR method for detecting Klebsiella pneumoniae according to claim 3, wherein the method for determining whether the sample to be detected is Klebsiella pneumoniae is a, b, c or d:
a. calcein color development method
Adding 1.0 mul of calcein indicator into a PSR reaction system; after the isothermal amplification reaction is finished, if the liquid of the reaction tube of the sample to be detected is yellow-green, the detection result of the sample is positive, wherein the detection result indicates that the klebsiella pneumoniae is detected; if the liquid of the reaction tube of the sample to be detected is orange red, the detection result is negative, and the detection result indicates that the Klebsiella pneumoniae is not detected;
b. pH indicator color development method
Adding 1.0 mu lpH indicator into the PSR reaction system; after the isothermal amplification reaction is finished, if the liquid of the reaction tube of the sample to be detected is yellow, the existence of the klebsiella pneumoniae in the sample to be detected is indicated; if the liquid of the reaction tube of the sample to be detected is red, the existence of Klebsiella pneumoniae in the sample to be detected is indicated; if the negative control tube is changed into yellow or the color of the positive control tube is not changed within 50 minutes, the detection result is invalid, and the detection is required to be carried out again;
c. turbidity method
Detecting a turbidity change curve of a PSR reaction product of the sample to be detected by a turbidity meter, wherein if the turbidity change curve of the PSR reaction product of the sample to be detected is an S-shaped reaction curve, Klebsiella pneumoniae exists in the sample to be detected, and if the turbidity change curve of the PSR reaction product of the sample to be detected does not form an obvious S-shaped reaction curve, the Klebsiella pneumoniae does not exist in the sample to be detected;
d. fluorescence method
The positive reaction appeared as: an S-shaped amplification curve appears in the reaction stage, and the dissolution curve is a single peak; the appearance of negative reactions was: no amplification curve exists in the reaction stage, and no peak appears in the dissolution curve; and (4) result judgment standard: under the premise that the positive control generates positive reaction and the negative control generates negative reaction, the sample to be detected generates an S-shaped amplification curve in the reaction stage, the dissolution curve is a single peak and is close to the position of the positive control single peak, the sample to be detected is judged to be positive, and the sample to be detected is negative otherwise; if the positive sample to be detected has negative reaction or the negative sample to be detected has positive reaction, the detection should be carried out again.
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