CN104946762B - Detect the kit of Friedlander's bacillus - Google Patents

Detect the kit of Friedlander's bacillus Download PDF

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CN104946762B
CN104946762B CN201510338356.0A CN201510338356A CN104946762B CN 104946762 B CN104946762 B CN 104946762B CN 201510338356 A CN201510338356 A CN 201510338356A CN 104946762 B CN104946762 B CN 104946762B
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friedlander
bacillus
kit
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primer
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CN104946762A (en
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杨君
易翔
黄茜
何犇
及思莹
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Zhongsheng Beikong Biological Science & Technology Co Ltd
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

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Abstract

The invention discloses a kind of kit for detecting Friedlander's bacillus, belong to PCR detection technique fields.Kit of the present invention includes being used for specific primer, the probe for detecting Friedlander's bacillus, its nucleotide sequence such as SEQ ID NO.1 3, and the target gene of detection is one section of sequence of phoE genes, and it has the nucleotide sequence shown in SEQ ID NO.4.The kit of the present invention has detection accurate, high sensitivity, high specificity, simple and rapid advantage, has good Samples detection ability, alternative traditional bacterium is separately cultured diagnostic method, and a kind of new quick detection kit is provided for Friedlander's bacillus.

Description

Detect the kit of Friedlander's bacillus
Technical field
The present invention relates to the PCR detection technique fields of strain, more particularly to for detecting the glimmering of Friedlander's bacillus Fluorescent Quantitative PCR primer pair and probe, the invention further relates to carry out determination of Klebsiella pneumoniae using the primer pair and probe Kit.
Background technology
Friedlander's bacillus belongs to Gram-negative bacteria, shaft-like, the pod membrane cladding that the polysaccharide for having a large amount of stickiness is formed.Lung Scorching Klebsiella is the important pathogen body of respiratory tract infection, often causes severe pneumonia, can also cause urethral infection, biliary tract sense The serious diseases such as dye, septicemia and purulent meningitis.Infect occurred in the debilitated patient being in hospital.Pathogen is often exhaled from above Road suction is inhaled, or human body is invaded by the artificial respirator of pollution, atomizer or various conduits.Friedlander's bacillus has turned into One of important pathogenic bacteria of inside-hospital infection, the first place of nosocomial infection is accounted in some countries.Therefore, clinically there is an urgent need to one Fast and accurately vitro detection means recognize types of spawn to kind, carry out targetedly individualized treatment.
Traditional bacteriologic test method is mainly entered by smear for microscopic examination and the method being separately cultured to Bacterial Physiological feature Row classification.The technological staff that the method for smear for microscopic examination needs to have wide experience is according to ne ar, arrangement mode and dyeability Tentative diagnosis is made, being separately cultured needs longer time to carry out result interpretation, is unfavorable for diagnosing cause of disease in time.Patient has The risk for sprawling that sb.'s illness took a turn for the worse.Fluorescent quantitative PCR technique not only realizes to be quantified to DNA/RNA templates, and with sensitive Degree and specificity height, the features such as multiple reaction, automaticity are high, stability is good, pollution-free, real-time and accurate, mesh can be realized Before be widely used to the fields such as molecular biology research and medical research.And traditional determination of Klebsiella pneumoniae method tool Have that time-consuming, recall rate is low, accuracy is low and the shortcomings that poor repeatability.
The content of the invention
It is an object of the invention to provide the kit for detecting Friedlander's bacillus.
To achieve the above object, the Friedlander's bacillus phoE gene order ratios that inventor has been reported ncbi database It is right, sequence homology analysis is carried out by BLAST softwares, finds specific conservative target sequence, it was found that nucleotides is protected completely Defending zone domain, for its nucleotide sequence as shown in SEQ ID NO.4, the target sequence is the genetic marker for detecting Friedlander's bacillus Thing.In addition, it will be appreciated by those skilled in the art that the specific fragment of the sequence can also be used as detection Friedlander's bacillus Genetic marker.
Further, the present invention also provides the primer for the above-mentioned target sequence of specific amplification, and coordinates with the primer The fluorescence probe used.Probe and primer can be designed using the grade softwares of Primer Express Version 3.It is logical Normal primer length is 15-30 base, and a primer sequence is identical with genetic marker sequence provided by the invention, and another is drawn Thing sequence is complementary with the genetic marker sequence；Usual Taqman probe lengths are 20-50 base, its sequence and above-mentioned heredity Label is identical or complementary, its 5 ' mark fluorescent group FAM, 3 ' mark quenching group BHQ-1.
In an embodiment of the invention, preferable primer sequence is：
Sense primer：5‘-GATGGGCTTTGTGGCTTCAA-3’
Anti-sense primer：5‘-CCAGCTTGTTCGCGTTCTTAT-3’.
Probe sequence is：
(FAM)5’-AGCGACGCAGGCAGCGGAA-3’(BHQ1)。
The invention provides above-mentioned specific primer pair and fluorescence probe to prepare detection Friedlander's bacillus kit In application.
The present invention provides a kind of real-time fluorescence quantitative PCR detection method of Friedlander's bacillus, including with sample total DNA For template, using above-mentioned genetic marker as target sequence, real time fluorescent quantitative is carried out using primer and probe provided by the invention PCR, while no template control and positive control are set up, according to amplification curve result of determination.
The present invention real-time fluorescence quantitative PCR amplification reaction system, when for 20 μ l reaction systems when, it is preferably configured to：
The response procedures of real-time fluorescence quantitative PCR of the present invention are：95 DEG C of pre-degeneration, 5min；15s are denatured through 95 DEG C again, 60 DEG C of annealing 60s, 35 circulations.
NTC controls (no template control) are must be set up during the sample of detection every time of the invention and POS compares (positive control), two Kind control plays a decisive role for result interpretation：
Effectively amplification：NTC (-) and POS (+)
Invalid amplification：NTC (+) and POS (+) prompting system pollution
Invalid amplification：NTC (-) and POS (-) prompting system mistake or reagent failure.
In the case of control effectively amplification, sample detection credible result, otherwise experiment needs to repeat；Two kinds in the detection When compareing effectively to expand, sample results criterion is as follows：
Sample of the Ct values less than or equal to 28 is positive findings；
Sample of the Ct values more than 35 is negative findings；
Sample of the Ct values between 28-35 needs to repeat, and repeats experiment such as Ct values and is still determined as positive amplification less than 28, It is determined as negative amplification more than 28.
The invention provides a kind of kit for detecting Friedlander's bacillus, it includes above-mentioned specific primer and matched somebody with somebody Close the Taqman probes that primer pair uses.
The specific primer pair and the sequence of fluorescence probe that kit of the present invention contains be：
Sense primer：5‘-GATGGGCTTTGTGGCTTCAA-3’
Anti-sense primer：5‘-CCAGCTTGTTCGCGTTCTTAT-3’.
Probe sequence is：
(FAM)5’-AGCGACGCAGGCAGCGGAA-3’(BHQ1)。
Kit provided by the invention, in addition to fluorescent quantitation reaction solution, negative template and positive template, the negative mould Plate is stoning sour water, and the positive template is Friedlander's bacillus standard positive template.
The invention provides the phoE gene conserved regions target sequence for detecting Friedlander's bacillus, and with the sequence The primer and probe of basic engineering is classified as, there is stronger specificity, Friedlander's bacillus is carried out using the primer and probe Fluorescent quantitative PCR detection method further simplify Friedlander's bacillus detection program, shorten detection cycle, for examining Friedlander's bacillus strain is surveyed, the positive rate of determination of Klebsiella pneumoniae as the means of detection clinical samples, can be improved.This The detection kit provided is invented, its reaction system contains above-mentioned primer, probe and negative positive control, and the kit pushes away Wide application is advantageous to quickly and accurately identify Friedlander's bacillus.
Brief description of the drawings
Fig. 1 is the fluorescent amplification curve of the Friedlander's bacillus different primers probe combinations in case study on implementation 1 of the present invention, Abscissa is threshold cycle number Ct values, and ordinate is relative fluorescence, wherein 1 represents primer combination of probe (1), 2 represent primer Probe combinations (2), 3 represent primer combination of probe (3), and 4 represent the negative control of three kinds of reaction systems.
Fig. 2 be case study on implementation 2 of the present invention in Friedlander's bacillus specific detection fluorescent amplification curve, horizontal seat Be designated as threshold cycle number Ct values, ordinate is relative fluorescence, wherein 1 represents Friedlander's bacillus, 2 represent VREF, Enterococcus faecalis, golden yellow glucose coccus, MRSE, Escherichia coli, Acinetobacter bauamnnii, enterobacter cloacae, haemolysis Portugal Grape coccus and negative control.
Fig. 3 be case study on implementation 3 of the present invention in Friedlander's bacillus sensitivity technique fluorescent amplification curve, horizontal seat Threshold cycle number Ct values are designated as, ordinate is relative fluorescence, wherein 1 represents 1 × 106The template of CFU/ml bacterium solutions, 2 represent 1 ×105The template of CFU/ml bacterium solutions, 3 represent 1 × 104The template of CFU/ml bacterium solutions, 4 represent 1 × 103The template of CFU/ml bacterium solutions, 5 represent 1 × 102The template of CFU/ml bacterium solutions, 6 represent negative control.
Fig. 4 is quantitative fluorescent PCR standard curve, and abscissa is the logarithm of initial template concentration, and ordinate is threshold cycle Number, Ct=-3.508lgX0+ 34.933, wherein Ct are threshold cycle number, X0For initial template concentration (copy/ml), R2= 0.9947。
Embodiment
Following examples further illustrate present disclosure, but should not be construed as limiting the invention.Without departing substantially from In the case of of the invention spirit and essence, the modifications or substitutions made to the inventive method, step or condition belong to the present invention Scope.
Unless otherwise specified, the conventional meanses that technological means used in embodiment is well known to those skilled in the art.
The foundation of the method for the fluorescence quantitative PCR detection Friedlander's bacillus of embodiment 1
1st, primer and probe designs
For the phoE gene conserved regions of Friedlander's bacillus, the present embodiment devises 3 pairs of primers and coordinated with it The probe used, 3 pairs of primers are respectively positioned on phoE genes, and its nucleotide sequence is as follows：
(1)
Sense primer：5‘-GATGGGCTTTGTGGCTTCAA-3’
Anti-sense primer：5‘-CCAGCTTGTTCGCGTTCTTAT-3’
Probe：5‘-AGCGACGCAGGCAGCGGAA-3’
(2)
Sense primer：5‘-CGATATGTTCCCGGAATTCG-3’
Anti-sense primer：5‘-GCTGGCGCGCTTGGT-3’
Probe：5‘-CGGCGACTCATCCGCTCAGACC-3’
(3)
Sense primer：5‘-GGCCGTTGGGAATCTGAATT-3’
Anti-sense primer：5‘-CGAACGCCAGACGAGTCTTT-3’
Probe：5‘-AACCGAGAGCGACTCCAGCCAGC-3’
5 ' end flag F AM fluorescent reporter groups, 3 ' end mark BHQ-1 fluorescence of described Taqman probe nucleotide sequences Quenching group.
2nd, the extracting genome DNA of Friedlander's bacillus
Boiling method extracts template DNA:It is 10 to take 1mL concentration5The Friedlander's bacillus bacterium solution of individual maxwell unit is placed in In 1.5mL sterile centrifugation tubes；Room temperature 10000g centrifuges 3min, abandons supernatant, add 80 μ L cell pyrolysis liquid (1%Triton, The EDTA of 10mM Tris-HCL, 1mM pH value 8.0), 6min, then room temperature 10000g centrifugation 3min are boiled, takes supernatant to place 4 DEG C Standby or -20 DEG C of storages.
3rd, fluorescent quantitative PCR
Taqman sonde method quantitative fluorescent PCRs are carried out according to following reaction system:
The template DNA of a certain amount of sample to be detected is added in PCR pipe, is positioned on quantitative real time PCR Instrument and is expanded Increase.Positive control and negative control be set respectively simultaneously, and positive reference substance is Friedlander's bacillus positive template, negative control Product are the deionized water of sterilizing.
4th, quantitative fluorescent PCR reaction condition is as follows：
The first step：95 DEG C of pre-degeneration, 5min；
Second step：95 DEG C of denaturation 15s, 60 DEG C of annealing 60s, totally 35 circulate.
5th, result interpretation：
Experimental result such as Fig. 1 shows that the fluorescent quantitative PCR Ct values of primer combination of probe (1) are 13, and amplification curve is Obvious S-shaped, it is positive amplification curve；Primer combination of probe (2), the fluorescent quantitative PCR Ct values of (3) are more than 28, for the moon Property amplification curve；Negative control is without amplification curve, therefore primer combination of probe (1) meets the requirements.Can be with from amplification curve Find out, the early stage of amplification, particularly near fluorescence critical value, curve co-insides are preferable.
6th, standard curve is drawn：
10 times of gradient dilutions are carried out according to the genomic DNA of Friedlander's bacillus type strain (CGMCC1.10617), it is dilute It is interpreted into 6 concentration and is expanded according to above-mentioned PCR reaction systems and program, after amplified reaction terminates, with initial template concentration The logarithm of (copy/ml) is abscissa, using threshold cycle number as ordinate, draws standard curve.Standard curve as shown in figure 4, Formula is Ct=-3.508lgX0+ 34.933, wherein Ct are threshold cycle number, X0For initial template concentration (copy/ml), R2= 0.9947。
The specificity verification of the fluorescence quantitative PCR detection Friedlander's bacillus method of embodiment 2
By Friedlander's bacillus, VREF, enterococcus faecalis, golden yellow glucose coccus, MRSE, large intestine bar Bacterium, Acinetobacter bauamnnii, enterobacter cloacae, MRSH bacterium solution are incubated overnight for 37 DEG C in LB culture mediums, extract bacterium solution Genomic DNA.Using the above-mentioned bacterium genomic DNA of extraction as template, sterile deionized water Taqman is carried out as negative control Sonde method quantitative fluorescent PCR reacts.As a result such as Fig. 2, Friedlander's bacillus template amplification curve are in S rows, and Ct values are 16, dung intestines It is coccus, enterococcus faecalis, golden yellow glucose coccus, MRSE, Escherichia coli, Acinetobacter bauamnnii, enterobacter cloacae, molten Blood staphylococcus and negative control are without amplification curve.As a result with being expected unanimously.
The sensitivity checking of the fluorescence quantitative PCR detection Friedlander's bacillus method of embodiment 3
Friedlander's bacillus is incubated overnight for 37 DEG C in LB culture mediums, carry out bacterium colony counting, then by concentration be 1 × 106CFU/ml bacterium solution carries out 10 times of gradient dilutions to 1 × 102CFU/ml.The genomic DNA of each concentration bacterium solution is extracted as mould Plate, sterile deionized water carry out the reaction of Taqman sonde methods quantitative fluorescent PCR as negative control.As a result such as Fig. 3 is shown, 1 generation Table 1 × 106The template of CFU/ml bacterium solutions, 2 represent 1 × 105The template of CFU/ml bacterium solutions, 3 represent 1 × 104The mould of CFU/ml bacterium solutions Plate, 4 represent 1 × 103The template of CFU/ml bacterium solutions, 5 represent 1 × 102The template of CFU/ml bacterium solutions, 20 μ under optimum reaction condition In l reaction systems, minimum detection limit 1 × 102The Ct values of CFU/ml genomic DNA starting template are about 25, therefore reaction follows Number of rings 35 can meet lowest detection requirement.From amplification curve as can be seen that S curve baseline is smooth, exponential region is obvious, slope compared with Greatly, these illustrate that the amplification of template under this condition is comparatively ideal.
Embodiment 4 detects the application of the kit of Friedlander's bacillus
In the present embodiment, inventor have collected the bacterium solution to be checked of 100 cultures, and base is extracted with the boiling method in embodiment 1 Fluorescence is carried out to the DNA of 100 bacterium solutions because of a group DNA, and using the primer combination of probe (1) obtained in the embodiment of the present invention 1 3,4,5 in quantitative PCR detection, detection method such as embodiment 1.Identified by " goldstandard " PCR sequencing PCR in this 100 samples Positive sample.
Testing result：PCR sequencing PCR, which identifies, contains 23 Friedlander's bacillus positive samples in 100 samples.Fluorescent quantitation PCR methods testing result is compared as follows with classical culture protocols testing result：
The comparison of the distinct methods positive rate of table 1
The distinct methods detection sensitivity of table 2 and specific comparison
Note：A kind of positive sample number/whole samples number of recall rate=method detection；
A kind of true positives sample number/goldstandard positive sample number of sensitivity=method detection；
A kind of true negative sample number/goldstandard negative sample number of specificity=method detection；
Can be drawn by data above, using the primer pair probe combinations (1) in embodiment 1 provided by the present invention and Fluorescence quantifying PCR method in embodiment 1 detects bacterium DNA to be checked, can substantially increase the spirit of determination of Klebsiella pneumoniae Sensitivity, specificity, and detection cycle is shortened compared with culture-based method, can be accurately and timely be provided with for diagnosing patient is tried hard to keep Card.
Although above the present invention is made to retouch in detail with general explanation, embodiment and experiment State, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art 's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, are belonged to claimed Scope.

Claims (8)

1. one kind is used for the genetic marker for detecting Friedlander's bacillus (K.peneumoniae), it is such as SEQ ID NO.4 institutes Show.
2. the specific primer pair for expanding genetic marker described in claim 1, its nucleotides sequence are classified as：
Sense primer：5‘-GATGGGCTTTGTGGCTTCAA-3’
Anti-sense primer：5‘-CCAGCTTGTTCGCGTTCTTAT-3’.
3. the fluorescence probe being used cooperatively with primer described in claim 2, its nucleotides sequence are classified as：5’- AGCGACGCAGGCAGCGGAA-3’。
4. a kind of kit for detecting Friedlander's bacillus, it is characterised in that contain the specific primer described in claim 2 Pair and claim 3 described in fluorescence probe.
5. kit as claimed in claim 4, it is characterised in that also including quantitative fluorescent PCR reaction reagent, positive reference substance And negative controls, the positive reference substance are the standard positive template of Friedlander's bacillus, negative controls go for sterilizing Ionized water.
6. kit as claimed in claim 4, it is characterised in that it uses real time fluorescence quantifying PCR method to be detected, its 20 μ L real-time fluorescence quantitative PCR reaction systems are：
7. according to any described kits of claim 4-6, it is characterised in that the working procedure of kit is：Pre-degeneration 95 ℃、5min；95 DEG C of denaturation 15s, 60 DEG C of annealing 60s, 35 circulate.
8. the fluorescence probe described in specific primer pair and claim 3 described in claim 2 is preparing detection kerekou pneumonia Application in primary Salmonella kit.
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101495647A (en) * 2005-06-02 2009-07-29 阿德万德克斯公司 Peptide nucleic acid probes for analysis of microorganisms
CN101892300A (en) * 2010-02-10 2010-11-24 广州华峰生物科技有限公司 Klebsiella pneumoniae detection kit and use method thereof
CN101928773A (en) * 2010-05-14 2010-12-29 中国人民解放军军事医学科学院卫生学环境医学研究所 Oligonucleotide primer for detecting common pathogenic bacteria by adopting fluorescent quantitation PCR (Rich Client Platform) technology, method thereof for detecting common pathogenic bacteria and application thereof
CN102367483A (en) * 2011-11-07 2012-03-07 中国农业大学 Specific primer pair for assisting identification of Klebsiella pneumoniae
CN103088144A (en) * 2013-01-30 2013-05-08 中国人民解放军军事医学科学院放射与辐射医学研究所 Detection kit of nucleic acid of klebsiella pneumoniae KPC (Klebsiella Pneumoniae Carbapenmase) type carbapenemases gene
CN103160574A (en) * 2011-12-09 2013-06-19 中山大学达安基因股份有限公司 Klebsiella pneumoniae nucleic acid detection kit (PCR-fluorescent probe method)
CN103642910A (en) * 2013-11-26 2014-03-19 车团结 Primer and probe for quantitative determination of klebsiella pneumonia, and application of primer and probe
CN104087676A (en) * 2014-07-18 2014-10-08 镜湖医院慈善会 Primer group and method for detecting four kinds of bacteria by means of multiple polymerase chain reaction (PCR)
CN104531887A (en) * 2015-01-15 2015-04-22 山东省动物疫病预防与控制中心 Identification method for Klebsiella pneumoniae and adopted primers

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101469327B (en) * 2007-12-25 2011-02-02 天津生物芯片技术有限责任公司 ITS sequence specific to Klebsiella pneumoniae pneumonia or subsp.ozaenae and use

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101495647A (en) * 2005-06-02 2009-07-29 阿德万德克斯公司 Peptide nucleic acid probes for analysis of microorganisms
CN101892300A (en) * 2010-02-10 2010-11-24 广州华峰生物科技有限公司 Klebsiella pneumoniae detection kit and use method thereof
CN101928773A (en) * 2010-05-14 2010-12-29 中国人民解放军军事医学科学院卫生学环境医学研究所 Oligonucleotide primer for detecting common pathogenic bacteria by adopting fluorescent quantitation PCR (Rich Client Platform) technology, method thereof for detecting common pathogenic bacteria and application thereof
CN102367483A (en) * 2011-11-07 2012-03-07 中国农业大学 Specific primer pair for assisting identification of Klebsiella pneumoniae
CN103160574A (en) * 2011-12-09 2013-06-19 中山大学达安基因股份有限公司 Klebsiella pneumoniae nucleic acid detection kit (PCR-fluorescent probe method)
CN103088144A (en) * 2013-01-30 2013-05-08 中国人民解放军军事医学科学院放射与辐射医学研究所 Detection kit of nucleic acid of klebsiella pneumoniae KPC (Klebsiella Pneumoniae Carbapenmase) type carbapenemases gene
CN103642910A (en) * 2013-11-26 2014-03-19 车团结 Primer and probe for quantitative determination of klebsiella pneumonia, and application of primer and probe
CN104087676A (en) * 2014-07-18 2014-10-08 镜湖医院慈善会 Primer group and method for detecting four kinds of bacteria by means of multiple polymerase chain reaction (PCR)
CN104531887A (en) * 2015-01-15 2015-04-22 山东省动物疫病预防与控制中心 Identification method for Klebsiella pneumoniae and adopted primers

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