CN112266969A - Target spot, primer, probe and kit for rapidly detecting klebsiella pneumoniae based on RAA fluorescence method - Google Patents
Target spot, primer, probe and kit for rapidly detecting klebsiella pneumoniae based on RAA fluorescence method Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- 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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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The application belongs to the technical field of molecular biology, and particularly relates to a Klebsiella pneumoniae gene highly conserved sequence, a detection primer probe and a kit thereof. A method for detecting Klebsiella pneumoniaeEnterobacter sakazakii) The nucleotide sequence of the target gene is shown as SEQ ID NO. 1. The primer probe designed based on the highly conserved sequence of the Klebsiella pneumoniae gene is suitable for RAA fluorescence detection, and can accurately detect the Klebsiella pneumoniae with the specificity of 100%. The present application also provides a method for fluorescence based on RAAThe kit for detecting the Klebsiella pneumoniae by the method can conveniently and accurately identify the existence of the Klebsiella pneumoniae, can be completed within 20min, and can be completed by carrying out isothermal amplification at 30-42 ℃.
Description
Technical Field
The application belongs to the technical field of molecular biology, and particularly relates to a target spot, a primer, a probe and a kit for rapidly detecting klebsiella pneumoniae based on an RAA fluorescence method.
Background
Klebsiella pneumoniae (Enterobacter sakazakii) belongs to Klebsiella of Enterobacteriaceae, and Friedlan der first isolated the Klebsiella pneumoniae from sputum of patients with lobar pneumonia in 1882. The Klebsiella pneumoniae is gram-negative bacillus, has no flagella and no spores, and can form capsules on nutrient-rich culture medium. Most strains have pili and have various main antigen components such as thalli antigen, capsular antigen, pili antigen and the like. The strain is facultative anaerobe, has low nutrition requirement, and can form relatively large, convex and gray-white mucus type bacterial colony on a culture medium.
Klebsiella pneumoniae is one of the most important conditional pathogenic bacteria, widely exists in micro-ecological environments such as water, soil and the like, is easy to be planted in respiratory tracts and intestinal tracts of inpatients, and can cause infection of various parts when the immunity of a body is low, wherein the infection comprises pneumonia, urinary tract infection, intra-abdominal infection, suppurative hepatic cyst, neonatal meningitis, bacteremia and the like. Klebsiella pneumoniae is the main pathogenic bacterium causing nosocomial pneumonia, and Klebsiella pneumoniae is the second approximate pathogen causing nosocomial infection in China, accounting for 9.03 percent, and can affect a plurality of lung lobes and cause lung pus and spleen, so the pneumonia is a clinical process with rapid progress, and an effective antibacterial treatment scheme is established only for a short time. Despite empirical treatment of patients, mortality rates exceed 50%. In recent years, the typical klebsiella pneumoniae strains have drug resistance and are in an ascending trend, so that the klebsiella pneumoniae strains are extremely troublesome in clinical treatment. Recently, it has been reported that a new highly toxic variant of klebsiella pneumoniae has emerged, which is clinically characterized as causing serious, life-threatening community-acquired infections in young healthy populations. If these strains also show a tendency to be resistant to drugs, clinical treatment will present a huge challenge. Therefore, there is a strong clinical need for a rapid, accurate and simple method for identifying pathogenic bacteria to assist in clinical selection of effective antibiotics to reduce mortality and the emergence of resistant strains.
Bacterial culture identification is a commonly used method for detecting klebsiella pneumoniae infection at present, and generally comprises the steps of smear, isolated culture, biochemical reaction identification, serological identification, enterotoxin determination and the like. Therefore, the method is tedious, time-consuming and labor-consuming, cross reaction exists in biochemical identification of homologous strains, and false negative is caused in a serological identification link due to low antibody titer, so that the conventional method cannot meet the requirements of clinical rapid identification and the requirements of drug selection. Molecular biology methods such as Polymerase Chain Reaction (PCR) and LAMP in isothermal nucleic acid amplification technology have been established and have been well applied to detection of Klebsiella pneumoniae, greatly shortening detection time. PCR technology such as real-time quantitative PCR makes a great breakthrough in the detection of Klebsiella pneumoniae due to its high specificity and sensitivity. But these methods require the use of complex instruments and well equipped laboratories. The LAMP method overcomes the defects that the PCR technology needs expensive equipment and instruments and the like, has the advantages of simple and convenient operation, simple result judgment and the like, and is applied to the detection of Klebsiella pneumoniae. However, the LAMP technique requires multiple pairs of primers and has high requirements for target genes and high false positives.
The application designs specific primers and probes based on recombinase-mediated isothermal nucleic acid amplification technology (RAA technology for short), and establishes a kit for rapidly detecting Klebsiella pneumoniae based on the RAA fluorescence method. The detection of the Klebsiella pneumoniae can be completed in about 20 minutes at 39 ℃, and the kit has the advantages of rapidness, sensitivity, simplicity and convenience in operation and the like, is suitable for field detection, and has important significance for the detection of the Klebsiella pneumoniae in food.
Disclosure of Invention
The first purpose of the application is to provide a target gene for detecting klebsiella pneumoniae (Enterobacter sakazakii), the target gene has high conservation, a probe for detecting a primer is designed by the target gene and has the characteristics of strong specificity, high sensitivity and good repeatability, the probe is further designed into a kit, and the kit has the advantages of rapidness, sensitivity, simplicity and convenience in operation and the like, is suitable for field detection, and has important significance for detecting the klebsiella pneumoniae in food.
In order to achieve the first object, the present application provides the following technical solutions:
a target gene for detecting Klebsiella pneumoniae (Enterobacter sakazakii) has a nucleotide sequence shown in SEQ ID NO. 1.
Further, the application also discloses primers for amplifying the target gene, wherein the primers comprise an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is as follows: CAGCGACGCAGGCAGCGGAAGTTTATAATAAG, the downstream primer nucleotide sequence is: TACCGCAGAATTCAGATTCCCAACGGCCATAG are provided.
Further, the application also discloses a probe for detecting the target gene, wherein the probe sequence is a 5 'terminal sequence/a fluorescent reporter group/A/THF/G/a quencher group/3' terminal sequence, and the probe sequence comprises the following steps: the nucleotide sequence of the 5' end is as follows: CTATGACAGCAAGGATGGCGATCAGACC, respectively; the nucleotide sequence of the 3' end is as follows: GCGTTTCGGTATTAAAG, respectively; the THF is at 1 base position.
Preferably, the fluorescent reporter group is selected from any one of FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy 5; the quenching group is selected from any one of TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL; most preferably, the fluorescence modifying group is FAM and the fluorescence quenching group is BHQ 1.
Preferably, the probe sequence is: CTATGACAGCAAGGATGGCGATCAGACCTACGTGCGTTTCGGTATTAAAG are provided.
Further, the application also discloses a kit for detecting Klebsiella pneumoniae based on the RAA fluorescence method, and the kit comprises an RAA basic fluorescence universal reaction reagent, a reaction buffer solution, a negative quality control product, a positive quality control product, a primer for amplifying the target gene and a probe for detecting the target gene.
Preferably, the primer adopts the upstream primer and the downstream primer, and the probe is the probe.
Preferably, the concentration of the upstream primer and the concentration of the downstream primer in the primers are independently 0.05-0.1 mmol/L; the concentration of the probe is 0.02-0.05 mmol/L.
Preferably, the reaction buffer comprises the following content components: Tris-HCl buffer solution with the concentration of 500mmol/L and the pH value of 7.4, MgAc with the concentration of 240mmol/L and PEG 10000 with the mass fraction of 10 percent.
Preferably, the positive quality control product contains genome DNA of Klebsiella pneumoniae; the concentration of the genome DNA of the Klebsiella pneumoniae is 1 × 104Copies/ul。
Preferably, the negative quality control product is ddH2O or purified water.
Further, the application also discloses application of the target gene, the primer or the probe in preparation of a kit for detecting Klebsiella pneumoniae based on an RAA fluorescence method.
Further, the application also discloses a method for detecting Klebsiella pneumoniae in food by using the kit, which comprises the following steps:
1) extracting DNA of a sample to be detected, and storing the obtained nucleic acid sample at-20 ℃ for later use;
2) switching on a power supply of a detection instrument RAA-F1620 for preheating, and setting reaction parameters, wherein the reaction parameters are set to 39 ℃, and the reaction time is as follows: 20 min;
3) adding 1 mu L of probe and 1 mu L of primer into 42.6 mu L of reaction buffer solution, mixing, adding into RAA basic fluorescence universal reaction reagent, and mixing; obtaining a reaction premixed solution;
4) fully mixing 5 mu L of the DNA extracting solution obtained in the step 1) with the reaction mixed solution obtained in the step 3), and putting the obtained reaction system into a detection instrument RAA-F1620 to detect a fluorescent signal;
5) according to the positive determination method in the RAA-F1620 detection apparatus, when the slope value K is not less than 20, the determination is positive. When the slope value K is less than 20, the judgment result is negative.
The primers and the probes provided by the application are suitable for RAA fluorescence detection, and can accurately detect Klebsiella pneumoniae, and the specificity reaches 100%.
The kit can conveniently, quickly and accurately identify the Klebsiella pneumoniae, is simple and convenient to operate, has short detection time, can be completed within 20min, does not need to unwind DNA through high temperature, and can complete detection only by carrying out isothermal amplification at 30-42 ℃.
The application discloses the detection method is quick, sensitive and efficient, can realize high throughput, and reduces detection time and detection cost, and research shows that the kit for quickly detecting klebsiella pneumoniae based on the RAA fluorescence method provided by the application has no cross reaction with salmonella, staphylococcus aureus, vibrio parahaemolyticus, streptococcus haemolyticus and shigella when detecting klebsiella pneumoniae, and has strong specificity which reaches 100%. The sensitivity is high, and the detection sensitivity per reaction reaches 10 Copies.
The kit for rapidly detecting klebsiella pneumoniae based on the RAA fluorescence method does not need large-scale instruments and equipment, and is suitable for field detection and large-scale screening.
Drawings
FIG. 1 shows the results of the sensitivity detection of plasmid DNA at different concentrations.
FIG. 2 shows the result of repetitive detection of Klebsiella pneumoniae plasmid DNA.
FIG. 3 shows the result of specific detection of Klebsiella pneumoniae.
Detailed Description
The application provides a primer, a probe and a kit for detecting Klebsiella pneumoniae based on an RAA fluorescence method, and the implementation method comprises the following steps:
1) designing a primer probe;
2) selecting a fluorescence modifying group and a fluorescence quenching group;
3) synthesizing a primer probe;
4) synthesizing positive plasmids;
5) preparing a positive plasmid standard substance;
6) RAA fluorescence detection experiment;
7) and extracting Klebsiella pneumoniae sample DNA for verification.
In the present application, the applicant screened out the highly conserved sequence of klebsiella pneumoniae as follows;
ATGATGATGGGCTTTGTGGCTTCAACAGCGACGCAGGCAGCGGAAGTTTATAATAAGAACGCGAACAAGCTGGATGTGTACGGCAAGATCAAAGCCATGCACTATTTCAGCGACTATGACAGCAAGGATGGCGATCAGACCTACGTGCGTTTCGGTATTAAAGGCGAAACGCAGATTAACGACGACCTGACCGGCTATGGCCGTTGGGAATCTGAATTCTGCGGTAACAAAACCGAGAGCGACTCCAGTCAGAAAACCCGTCTGGCGTTC。
taking a highly conserved sequence as a detection target gene, synthesizing positive plasmids and designing primer probes; designing a primer, wherein the primer design comprises an upstream primer and a downstream primer; DNA plasmids were synthesized according to the above sequence of Competition Biotechnology engineering (Shanghai) Ltd. and the plasmid size was 270 bp.
1. Primer Probe design
Designing by adopting an RAA technical primer and probe design principle, and determining an upstream primer CAGCGACGCAGGCAGCGGAAGTTTATAATAAG through screening and evaluation; a downstream primer TACCGCAGAATTCAGATTCCCAACGGCCATAG; the probe sequence was CTATGACAGCAAGGATGGCGATCAGACCTACGTGCGTTTCGGTATTAAAG.
2. Selection of fluorescence modifying group and fluorescence quenching group
According to the experimental instrument, the RAA-F1620 fluorescence gene detector produced by Wuxi-Tian-bioscience instruments, Inc. is adopted, and the detected fluorescence is FAM fluorescence, so that a fluorescence modifying group is selected as FAM, and a fluorescence quenching group is selected as BHQ 1;
the fluorescent modifying group can also be selected from HEX, TET, JOE, VIC, ROX, Cy3 or Cy5 according to the fluorescence detection performance of the instrument. The fluorescence quenching group is selected from TAMRA, Eclipse, BHQ2, BHQ3 or DABCYL. However, the preferred fluorescence modifying group is FAM, and the preferred fluorescence quenching group is BHQ 1.
3. The method for modifying the probe preferably includes: the fluorescent reporter group is modified on the position of 29bp away from the 5' end base number of the probe sequence; the fluorescence quenching group is modified on the position of 18bp away from the 3' end base number of the probe sequence, and the modified probe is as follows: CTATGACAGCAAGGATGGCGATCAGACC/i6 FAMdT/A/THF/G/iBHQdT/GCGTTTCGGTATTAAAG.
4. The primer probe and the plasmid were synthesized by the firm Biotech engineering (Shanghai) Ltd.
5. Preparation of plasmid-positive Standard
Culturing and extracting recombinant plasmid by transferring Escherichia coli, measuring concentration with ultramicro ultraviolet spectrophotometer, calculating copy number, diluting according to concentration gradient to obtain 1.0 × 100copies/ul-1.0×1010Copies/ul for standby.
5. Make up kit
A kit for rapidly detecting Klebsiella pneumoniae based on an RAA fluorescence method comprises an RAA basic fluorescence universal reaction reagent, a reaction buffer solution, a positive quality control product, a negative quality control product, a primer and a probe; the concentration of the upstream primer and the concentration of the downstream primer are independently preferably 0.05-0.1 mmol/L, and more preferably 0.08 mmol/L. The concentration of the probe is preferably 0.02 to 0.05mmol/L, and more preferably 0.04 mmol/L.
The kit comprises an RAA basic fluorescence universal reaction reagent. The RAA basic fluorescence universal reaction reagent is preferably freeze-dried powder subjected to low-temperature freeze drying. In the embodiment of the application, the RAA basic fluorescence universal reaction reagent is purchased from Jiangsu Qitian gene biotechnology limited, and the product number is F01006. The reaction specification of the RAA-based fluorescent universal reaction reagent is 50. mu.L, and a reaction buffer solution is applied for re-dissolution before use.
The reaction buffer for redissolving the RAA-based fluorescent universal reaction reagent preferably comprises the following components in percentage by weight: Tris-HCl (PH7.4) buffer solution with the concentration of 500mmol/L, MgAc with the concentration of 240mmol/L and PEG 10000 with the mass fraction of 10 percent. In the present application, the concentrations of the Tris-HCl buffer, the MgAc and the PEG 10000 are final concentrations. The sources of the Tris-HCl buffer solution, the MgAc and the PEG 6000 are not particularly limited, and the conventional commercial products can be adopted. Tris-HCl, and PEG 6000 as described in the examples of this application were obtained from Sigma-Aldrich and MgAc from Shanghai test, national medicine.
The kit comprises a positive quality control product. The positive quality control product preferably contains genome DNA of Klebsiella pneumoniae. The concentration of the genomic DNA of the Klebsiella pneumoniae is preferably 1 × 106Copies/ul. The positive quality control substances in the embodiment of the application are cultured and extracted by transferring recombinant plasmids into escherichia coli.
The kit comprises a negative quality control product. The negative quality control product is preferably ddH2O or purified water.
In the present application, the method for using the kit for detecting klebsiella pneumoniae by the RAA fluorescence method according to the above-described embodiment preferably includes the following steps:
(1) the DNA of a sample to be detected (suspected Klebsiella pneumoniae) is extracted by adopting a commercially available bacterial nucleic acid extraction reagent according to the instruction. Storing the obtained nucleic acid sample at-20 deg.C;
(2) switching on a power supply of a detection instrument RAA-F1620 for preheating, and setting reaction parameters, wherein the reaction parameters are set to 39 ℃, and the reaction time is as follows: and 20 min.
(3) Adding 1 mu L of probe and 1 mu L of primer into 42.6 mu L of reaction buffer solution, mixing, adding into RAA basic fluorescence universal reaction reagent, and mixing; obtaining a reaction premixed solution;
(4) fully mixing 5 mu L of the DNA extracting solution obtained in the step (1) with the reaction mixed solution obtained in the step (3), and putting the obtained reaction system into a detection instrument RAA-F1620 to detect a fluorescent signal;
(5) according to the positive determination method in the RAA-F1620 detection apparatus, when the slope value K is not less than 20, the determination is positive. When the slope value K is less than 20, the judgment result is negative.
The technical solutions in the present application will be clearly and completely described below with reference to the embodiments in the present application. It is to be understood that the embodiments described are only a few embodiments of the present application and not all 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 application.
Example 1 sensitivity test
The composition of the kit is shown in table 1:
TABLE 1 kit Components Table
The prepared recombinant plasmid working standard comprises the following components:
working standard 1, containing 1.0 × 106Plasmid DNA segment of Copies/ul Klebsiella pneumoniae.
Working standard 2, containing 1.0 × 105Plasmid DNA segment of Copies/ul Klebsiella pneumoniae.
Working standard 3, containing 1.0 × 104Plasmid DNA segment of Copies/ul Klebsiella pneumoniae.
Working standard 4, containing 1.0 × 103Plasmid DNA segment of Copies/ul Klebsiella pneumoniae.
Working standard 5, containing 1.0 × 102Plasmid DNA segment of Copies/ul Klebsiella pneumoniae.
Working standard 6, containing 1.0 × 101Plasmid DNA segment of Copies/ul Klebsiella pneumoniae.
The implementation method comprises the following steps:
1. preparation of reaction buffer
And (3) sucking 301 mu L of reaction buffer solution from a reaction buffer solution tube in the kit, adding the reaction buffer solution into a prepared 1.5ml PE tube, adding 16 mu L of a mixture of the probe and the primer (the concentration of the probe is 0.02mmol/, the concentration of the primer is 0.05mmol/L), and fully mixing to obtain the uniformly mixed reaction buffer solution.
2. Re-dissolving RAA fluorescent basic reaction reagent
Preparing 7 RAA fluorescence basic reaction reagents, sucking 45 mu L of the reaction buffer solution uniformly mixed in the step 1, and respectively adding the reaction buffer solution into the prepared 7 RAA fluorescence basic reaction reagent tubes to fully dissolve and uniformly mix the freeze-dried powder to form an RAA reaction system.
3. Sample application reaction
Respectively adding 1 mu L of negative quality control material, 1 mu L of standard work 6, 1 mu L of standard work 5, 1 mu L of standard work 4, 1 mu L of standard work 3 and 1 mu L of standard work 2 into the 7 prepared RAA fluorescence basic reaction reagent test tubes, and fully mixing each reaction tube after adding the sample, wherein the total volume of each reaction tube is 50 mu L.
The reaction tube was placed in a RAA-F1620 fluorescence detector, and the reaction temperature was set at 39 ℃ for 20 minutes. The detection results are shown in FIG. 1. The result shows that all standard works are amplified within 25 minutes, and the lowest sensitivity can reach 1.0 multiplied by 101Copies/ul, which shows that the sensitivity of the application is high.
Example 2 repeatability experiments
The primer probe and the positive quality control substance sequence are the same as those in example 1.
The composition of the kit is shown in table example 1:
1. preparing a reaction buffer solution:
173 mul of reaction buffer solution is sucked from a reaction buffer solution tube in the kit and added into a prepared 1.5ml PE tube, 8 mul of mixture of the probe and the primer (the concentration of the probe is 0.05mmol/L and the concentration of the primer is 0.1mmol/L) is added, and the mixture is fully and evenly mixed to obtain the evenly mixed reaction buffer solution.
2. Re-dissolving RAA fluorescent basic reaction reagent
Preparing 4 RAA fluorescence basic reaction reagents, sucking 45 mu L of the reaction buffer solution uniformly mixed in the step 1 each time, respectively adding the reaction buffer solution into the prepared 4 RAA fluorescence basic reaction reagent tubes, fully dissolving and uniformly mixing the freeze-dried powder to form an RAA reaction system, and marking.
3. Sample application reaction
Respectively adding 1 mu L of negative quality control materials into the 4 prepared RAA fluorescent basic reaction reagent test tubes, and respectively adding 1 mu L of Klebsiella pneumoniae nucleic acid samples into the other 3 reaction tubes; the tube cover is covered after adding one sample. After the sample is added, each reaction tube is fully mixed, and the total volume of each reaction tube is 50 mu L.
The 4 uniformly mixed reaction tubes are placed into an RAA-F1620 fluorescence detector, the reaction temperature is set to be 39 ℃, and the reaction time is set to be 20 minutes. The detection results are shown in FIG. 2. The results show that the amplification reaction is highly reproducible.
Example 3 specificity test
The primer probe and the positive quality control substance sequence are the same as those in example 1.
The composition of the kit is shown in table 1 of example 1:
in the specificity experiment, nucleic acid samples of Klebsiella pneumoniae, salmonella, staphylococcus aureus, vibrio parahaemolyticus, streptococcus haemolyticus and Shigella are obtained by extracting original strains after amplification culture.
The implementation method comprises the following steps:
1. preparation of reaction buffer
Sample DNA reaction buffer preparation: and (3) sucking 301 mu L of reaction buffer solution from a reaction buffer solution tube in the kit, adding the reaction buffer solution into a prepared 1.5ml PE tube, adding 16 mu L of a mixture of the probe and the primer (the concentration of the probe is 0.04mmol/, the concentration of the primer is 0.08mmol/L), and fully mixing to obtain the uniformly mixed reaction buffer solution.
2. Re-dissolving RAA fluorescent basic reaction reagent
Preparing 7 RAA fluorescence basic reaction reagents, sucking 45 mu L of the reaction buffer solution uniformly mixed in the step 1 each time, respectively adding the reaction buffer solution into the prepared 7 RAA fluorescence basic reaction reagent tubes, fully dissolving and uniformly mixing the freeze-dried powder to form an RAA reaction system, and marking.
3. Sample application reaction
Respectively adding 5 mu L of negative quality control product, 5 mu L of Klebsiella pneumoniae nucleic acid, 5 mu L of salmonella nucleic acid, 5 mu L of staphylococcus aureus nucleic acid, 5 mu L of vibrio parahaemolyticus nucleic acid, 5 mu L of streptococcus haemolyticus nucleic acid and 5 mu L of shigella nucleic acid into the 7 prepared RAA fluorescent basic reaction reagent test tubes; the tube cover is covered after adding one sample. After the sample is added, each reaction tube is fully mixed, and the total volume of each reaction tube is 50 mu L.
And (3) putting the uniformly mixed 7 reaction tubes into an RAA-F1620 fluorescence detector, setting the reaction temperature to be 39 ℃, and reacting for 20 minutes. The results are shown in FIG. 3. The result shows that only Klebsiella pneumoniae nucleic acid is amplified and is positive, and other samples such as salmonella nucleic acid, staphylococcus aureus nucleic acid, vibrio parahaemolyticus nucleic acid, streptococcus haemolyticus nucleic acid, shigella nucleic acid and negative quality control products are not amplified and are negative. The detection method provided by the application is proved to be strong in specificity.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present disclosure, including any person skilled in the art, having the benefit of the present disclosure. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the application. Thus, the present application 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.
Sequence listing
<110> Hangzhou customs technology center
JIANGSU QITIAN GENE BIOTECHNOLOGY Co.,Ltd.
<120> target spot, primer, probe and kit for rapidly detecting Klebsiella pneumoniae based on RAA fluorescence method
<141> 2020-10-28
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 270
<212> DNA
<213> Klebsiella pneumoniae (Klebsiella pneumoniae)
<400> 1
atgatgatgg gctttgtggc ttcaacagcg acgcaggcag cggaagttta taataagaac 60
gcgaacaagc tggatgtgta cggcaagatc aaagccatgc actatttcag cgactatgac 120
agcaaggatg gcgatcagac ctacgtgcgt ttcggtatta aaggcgaaac gcagattaac 180
gacgacctga ccggctatgg ccgttgggaa tctgaattct gcggtaacaa aaccgagagc 240
gactccagtc agaaaacccg tctggcgttc 270
<210> 2
<211> 32
<212> DNA
<213> Artificial (artificial)
<400> 2
cagcgacgca ggcagcggaa gtttataata ag 32
<210> 3
<211> 32
<212> DNA
<213> Artificial (artificial)
<400> 3
taccgcagaa ttcagattcc caacggccat ag 32
<210> 4
<211> 50
<212> DNA
<213> Artificial (artificial)
<400> 4
ctatgacagc aaggatggcg atcagaccta cgtgcgtttc ggtattaaag 50
Claims (10)
1. A method for detecting Klebsiella pneumoniaeEnterobacter sakazakii) The target gene is characterized in that the nucleotide sequence of the target gene is shown as SEQ ID NO. 1.
2. The primer for amplifying the target gene of claim 1, wherein the primer comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is: CAGCGACGCAGGCAGCGGAAGTTTATAATAAG, the downstream primer nucleotide sequence is: TACCGCAGAATTCAGATTCCCAACGGCCATAG are provided.
3. The probe for detecting the target gene as claimed in claim 1, wherein the probe sequence is 5 'terminal sequence/fluorescent reporter group/A/THF/G/quencher group/3' terminal sequence, wherein: the nucleotide sequence of the 5' end is as follows: CTATGACAGCAAGGATGGCGATCAGACC, respectively; the nucleotide sequence of the 3' end is as follows: GCGTTTCGGTATTAAAG, respectively; the THF is at 1 base position; preferably, the fluorescent reporter group is selected from any one of FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy 5; the quenching group is selected from any one of TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL; most preferably, the fluorescence modifying group is FAM and the fluorescence quenching group is BHQ 1.
4. A probe for detecting a target gene of claim 1, wherein the probe sequence is: CTATGACAGCAAGGATGGCGATCAGACCTACGTGCGTTTCGGTATTAAAG are provided.
5. A kit for detecting Klebsiella pneumoniae based on an RAA fluorescence method is characterized by comprising an RAA basic fluorescence universal reaction reagent, a reaction buffer solution, a negative quality control product, a positive quality control product, a primer for amplifying the target gene of claim 1 and a probe for detecting the target gene of claim 1.
6. The kit according to claim 5, wherein the primer is the primer according to claim 2, and the probe is the probe according to claim 3 or 4.
7. The kit according to claim 5 or 6, wherein the concentration of the upstream primer and the concentration of the downstream primer in the primers are independently 0.05-0.1 mmol/L; the concentration of the probe is 0.02-0.05 mmol/L.
8. The kit according to claim 5 or 6, wherein the reaction buffer comprises the following content components: Tris-HCl buffer solution with the concentration of 500mmol/L and the pH value of 7.4, MgAc with the concentration of 240mmol/L and PEG 10000 with the mass fraction of 10 percent; the positive quality control product contains genome DNA of Klebsiella pneumoniae; the concentration of the genome DNA of the Klebsiella pneumoniae is 1×104Copies/ul; the negative quality control product is ddH2O or purified water.
9. Use of the target gene of claim 1, the primer of claim 2, or the probe of claim 3 or 4 in the preparation of a kit for detecting klebsiella pneumoniae based on a RAA fluorescence method.
10. A method for detecting klebsiella pneumoniae in food using the kit according to any one of claims 5 to 8, the method comprising the steps of:
1) extracting DNA of a sample to be detected, and storing the obtained nucleic acid sample at-20 ℃ for later use;
2) switching on a power supply of a detection instrument RAA-F1620 for preheating, and setting reaction parameters, wherein the reaction parameters are set to 39 ℃, and the reaction time is as follows: 20 min;
3) adding 1 mu L of probe and 1 mu L of primer into 42.6 mu L of reaction buffer solution, mixing, adding into RAA basic fluorescence universal reaction reagent, and mixing; obtaining a reaction premixed solution;
4) fully mixing 5 mu L of the DNA extracting solution obtained in the step 1) with the reaction mixed solution obtained in the step 3), and putting the obtained reaction system into a detection instrument RAA-F1620 to detect a fluorescent signal;
5) according to the positive determination method in the RAA-F1620 detection instrument, the determination is positive when the slope value K is larger than or equal to 20, and the determination is negative when the slope value K is smaller than 20.
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| CN202011237896.7A CN112266969A (en) | 2020-11-09 | 2020-11-09 | Target spot, primer, probe and kit for rapidly detecting klebsiella pneumoniae based on RAA fluorescence method |
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