CN106868160A - Primer and its application of various diarrhoea pathogenic bacterias are detected simultaneously - Google Patents

Primer and its application of various diarrhoea pathogenic bacterias are detected simultaneously Download PDF

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Publication number
CN106868160A
CN106868160A CN201710167845.3A CN201710167845A CN106868160A CN 106868160 A CN106868160 A CN 106868160A CN 201710167845 A CN201710167845 A CN 201710167845A CN 106868160 A CN106868160 A CN 106868160A
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seq
probe
primer
primers
pair
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任绪义
虞闰六
杨燚超
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Hangzhou Da Diagnostics Center Co Ltd
Hangzhou Dian Biotechnology Co Ltd
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Hangzhou Da Diagnostics Center Co Ltd
Hangzhou Dian Biotechnology Co Ltd
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses the primer and its application that detect various diarrhoea pathogenic bacterias simultaneously, belong to pathogen field of nucleic acid detection.The invention discloses SEQ ID NO:Primer and probe shown in 1~39, can quickly and accurately detect staphylococcus aureus, shigella dysenteriae, vibrio parahaemolytious, YE, Clostridium difficile bacillus, Plesiomonas, Listeria, EHEC O157：H7, Aeromonas, detection of Salmonella, C.perfringens, Bacillus cercus and campylobacter jejuni, are that Clinical microorganism detection band carrys out great convenience.

Description

Primer and its application of various diarrhoea pathogenic bacterias are detected simultaneously
Technical field
The invention belongs to the pathogenic microorganism examination field, and in particular to a kind of while detecting the reagent of various diarrhoea pathogenic bacterias Box and application.
Background technology
Infectious diarrhea is current global important public health problem, and regional less than 5 years old children are annual for sub-, non-, drawing Because of diarrhoea, died is about 5,000,000, and year morbidity number is about 7.5 hundred million~1,000,000,000 person-times.Infectious diarrhea pathogen mainly has sand Door bacterium, shigella dysenteriae, campylobacter jejuni, enteropathogenic E. Coli etc..At present, mainly there is conventional point for the detection of diarrhoeal diseases substance From culture and biochemical identification, immunological method, molecular biology method.Normal isolation culture is cumbersome, time-consuming, man's activity It is larger, easily cause missing inspection；Immunological method need not be complicated instrument, simple to operate, but antigenicity due to pathogen and Virulence factor is not necessarily related, causes cause of disease to be difficult to determine, immunological method is also relied on the pure bacterium of higher concentration, increases bacterium process ratio Relatively time consuming, can not cultivate bacterium for some can not detect；Molecular biology method mainly have probe hybridize, it is multiple in real time PCR and genetic chip, substantially increase the speed and sensitiveness of detection.
So-called Real-Time Fluorescent Quantitative PCR Technique, refers to add fluorophor in PCR reaction systems, using fluorescence signal The accumulation whole PCR processes of real-time monitoring, the method for carrying out quantitative analysis to unknown template finally by standard curve.The technology is not Only realize and DNA/RNA templates quantified, and with sensitivity and specificity it is high, can realize multiple reaction, automation journey The features such as degree is high, pollution-free, with low cost.Existing various kits with quantitative PCR technique are applied to clinical disease at present Related pathogen detection, Genotyping, mutation research etc..What is applied in this research is taqman fluorescence probes as fluorescent base Group, adds a specific fluorescence probe while pair of primers is added when PCR is expanded, the probe is an oligonucleotides, Two ends one reporter fluorescence group of mark and a quenching fluorescence group respectively.When probe is complete, the fluorescence of reporter group transmitting Signal is quenched group absorptions；When PCR is expanded, the 5'-3' 5 prime excision enzyme activities of Taq enzyme degrade probe digestion, make reporter fluorescence Group and quenching fluorescence group are separated, so that fluorescence monitoring system can receive fluorescence signal, i.e., often expand a DNA, just There is a fluorescence molecule to be formed, the accumulation and PCR primer for realizing fluorescence signal form Complete Synchronization.
The content of the invention
It is an object of the invention to provide a kind of while expand the primer of various diarrhoea pathogenic bacterias, to solve to grasp in the prior art Make cumbersome, time-consuming, man's activity is larger, easily cause the problem of missing inspection.
The technical problem also to be solved of the invention is to provide including above-mentioned while expanding the primer of various diarrhoea pathogenic bacterias Kit.
The technical problem finally to be solved of the invention is, above-mentioned while expanding answering for the kit of various diarrhoea pathogenic bacterias With.
In order to solve the above technical problems, the present invention is adopted the following technical scheme that：
The primer of various diarrhoea pathogenic bacterias is detected simultaneously, it is characterised in that including following five groups of primers：
First group of amplification Staphylococcus aureus nuc genes, Shigella Gene ipaH Using, tlh gene of vibrio parahaemolyticus primer pair, Including following three pairs of primers and probe：
Detect the primer pair and probe of staphylococcus aureus nuc genes：
F1(SEQ ID NO.：1)：ATCCTAAAAAAGGTGTAGAG,
R1(SEQ ID NO.：2)：TATCAGTTCTTTGACCTTTG,
P1(SEQ ID NO.：3)：FAM-ATATGGTCCTGAAGCAAGTGCA-MGB；
Detect the primer and probe of Shigella Gene ipaH Using：
F2(SEQ ID NO.：4)：ATAAAGTCAGAACTCTCC,
R2(SEQ ID NO.：5)：AACGCATTTCCTTCACGG,
P2(SEQ ID NO.：6)：VIC-ATGAGATAGAAGTCTACCTGGCCTTCC-TAMRA；
Detect the primer and probe of tlh gene of vibrio parahaemolyticus：
F3(SEQ ID NO.：7)：TCAACCGCTCATCGTCTGT,
R3(SEQ ID NO.：8)：CTGCGACATAGCGGTGAGT,
P3(SEQ ID NO.：9)：CY5-CACCCACGCATTGCGCTCTGAGTGT-BQ2；
Second group of amplification YE ail genes, clostridium difficile tpi genes, Plesiomonas shigelloides The primer and probe of hugA genes, including following three pairs of primers and probe：
Detect the primer and probe of YE ail genes：
F4(SEQ ID NO.：10)：TAATAGGTTCGTTTGCTTATACC,
R4(SEQ ID NO.：11)：GCGGAAAGATGGCCCCATTGTTA,
P4(SEQ ID NO.：12)：FAM-CGATTTCTTCTATGGCAGTAATAAGT–TAMRA；
Detect the primer and probe of clostridium difficile tpi genes：
F5(SEQ ID NO.：13)：ACTGATGAAACTGTAAACA,
R5(SEQ ID NO.：14)：AACATCTTTAGTTTTTCCA,
P5(SEQ ID NO.：15)：HEX-CCCAATATTATGTGTTGGAGAAACTTTA-T AMRA；
Detection Plesiomonas shigelloides hugA genes：
F6(SEQ ID NO.：16)：TGACTGCTTTGAGCGCGCC,
R6(SEQ ID NO.：17)：AATCACAAGATGAATTGCC,
P6(SEQ ID NO.：18)：CY5-AGTGGTGTAGAGATCCACCAAGGTGTAG-BQ2；
3rd group of amplification Listeria hlyA genes, EHEC O157:H7rfbE genes, vibrio parahaemolytious tlh bases The primer and probe of cause, including following three pairs of primers and probe：
Expand the primer and probe of Listeria hlyA genes：
F7(SEQ ID NO.：19)：CAGGTGATGTAGAACTGACAAA,
R7(SEQ ID NO.：20)：GCACCTTTTTTCAAAATATCTC,
P7(SEQ ID NO.：21)：FAM-AAAGCCGTAATTTACGGTGGCTCCG-TAMRA；
Amplification EHEC O157:H7rfbE gene primers and probe：
F8(SEQ ID NO.：22)：TTTCGTTGATTCAGATAATG,
R8(SEQ ID NO.：23)：AAATTTCTACTTTTGGCCAG,
P8(SEQ ID NO.：24)：HEX-TAGTGACATAGAACAAAAAATCACTAA-TA MRA；
Amplification tlh gene of vibrio parahaemolyticus primer and probe：
F3(SEQ ID NO.：7)：TCAACCGCTCATCGTCTGT,
R3(SEQ ID NO.：8)：CTGCGACATAGCGGTGAGT,
P3(SEQ ID NO.：9)：CY5-CACCCACGCATTGCGCTCTGAGTGT-BQ2；
4th group of amplification Aeromonas gcat gene, detection of Salmonella invA genes, C.perfringens cpa gene primers pair and Probe, including following three pairs of primers and probe：
Expand the primer and probe of Aeromonas gcat genes：
F9(SEQ ID NO.：25)：AAGGCGGTGCCACTGCCGT,
R9(SEQ ID NO.：26)：TCCGGCTTGAAGCTGTCTT,
P9(SEQ ID NO.：27)：FAM-TATCAGGTCATCAACAACCTGGACT-TAMRA；
Expand the primer and probe of detection of Salmonella invA genes：
F10(SEQ ID NO.：28)：TATTGGCGATAGCCTGGCGG,
R10(SEQ ID NO.：29)：TCGGCATCAATACTCATCTG,
P10(SEQ ID NO.：30)：Vic-AGTTTATCGTTATTACCAAAGGT-MGB；
Expand the primer and probe of C.perfringens cpa genes：
F11(SEQ ID NO.：31)：AGGGAAATCAATATACTATA,
R11(SEQ ID NO.：32)：TGAGAGTTAGCTAGAGTTAC,
P11(SEQ ID NO.：33)：CY5-AGTCATGCTAGCATGAGTCATAGTT-BQ2；
5th group of amplification staphylococcus aureus nuc gene, Bacillus cercus cesB genes, campylobacter jejuni mapA Primer and probe, including following three pairs of primers and probe：
Expand the primer and probe of staphylococcus aureus nuc genes：
F1(SEQ ID NO.：1)：ATCCTAAAAAAGGTGTAGAG,
R1(SEQ ID NO.：2)：TATCAGTTCTTTGACCTTTG,
P1(SEQ ID NO.：3)：FAM-ATATGGTCCTGAAGCAAGTGCA-MGB；
Expand the primer and probe of Bacillus cercus cesB genes：
F12(SEQ ID NO.：34)：CTGATGGGTTTAATGTAATGAA,
R12(SEQ ID NO.：35)：GTCTGACGTAATTCTGCTAATA,
P12(SEQ ID NO.：36)：VIC-ATTATAGTCGGAGTAGCAAGGGATAA–MGB；
The primer and probe of amplification campylobacter jejuni mapA：
F13(SEQ ID NO.：37)：TGCTCCATAAGATATAAATT,
R13(SEQ ID NO.：38)：TTTATACTTATTTAAAACAA,
P13(SEQ ID NO.：39)：CY5-CATAAGGTGAATTTTGATCGTTATTGT-BQ2.
The kit of various diarrhoea pathogenic bacterias, including following reagent are detected simultaneously：
(1) PCR reaction solutions:10*PCR buffer solutions, Q buffer solutions, archaeal dna polymerase, Mg2+And dNTPs, the Q buffer solutions bag Include following component：Glycine betaine 2.7M, DMSO6.7%, DTT6.7mM, BSA0.055mg/ml；
(2) primer and probe mixed liquor:By SEQ ID NO.：Nucleotide sequence shown in 1~39 is according to claim 1 point Group, final concentration of 1 μm of ol/L of every kind of primer；
(3) positive control：Containing diarrhoea pathogenic bacterial genomes DNA, wherein, each diarrhoea pathogenic bacterial genomes DNA concentration is 10ng/ μ L；
(4) negative control：Genome of E.coli DNA, concentration is 100ng/ μ L.
It is above-mentioned while detecting the kit of various diarrhoea pathogenic bacterias applying of the invention in diarrhoea pathogenic bacteria is detected Within protection domain.
Wherein, the diarrhoea pathogenic bacteria includes：Staphylococcus aureus, shigella dysenteriae, vibrio parahaemolytious, enterocolitis Ademilson bacterium, Clostridium difficile bacillus, Plesiomonas, Listeria, EHEC O157：H7, Aeromonas, detection of Salmonella, aerogenesis Application in capsular clostridium, Bacillus cercus and campylobacter jejuni.
Beneficial effect：
The invention discloses a kind of while detect primer and its application of various diarrhoea pathogenic bacterias, by 5 groups of fluorescent quantitations PCR can detect staphylococcus aureus, shigella dysenteriae, vibrio parahaemolytious, YE, Clostridium difficile bar Bacterium, Plesiomonas, Listeria, EHEC O157：H7, Aeromonas, detection of Salmonella, C.perfringens, wax-like gemma Bacillus and campylobacter jejuni totally 13 kinds of diarrhoea pathogenic bacterias.The detection of bacterium is limited to 10~104Cfu/ml, sensitivity is higher.
Brief description of the drawings
The sensitivity of Fig. 1 detection staphylococcus aureus primers is 10cfu/ml.
The sensitivity of Fig. 2 detection shigella dysenteriaes is 10cfu/ml.
The sensitivity of Fig. 3 detection vibrio parahaemolytious is 102cfu/ml。
The sensitivity of Fig. 4 detection YEs is 10cfu/ml.
The sensitivity of Fig. 5 detection clostridium difficiles is 104cfu/ml
The sensitivity of Fig. 6 detection Plesiomonas shigelloides is 102cfu/ml
The sensitivity of Fig. 7 detection Listeria monocytogenes is 104cfu/ml
Fig. 8 EHECs O157：The sensitivity of H7 is 103cfu/ml
The sensitivity of Fig. 9 detection Aeromonas is 103cfu/ml
The sensitivity of Figure 10 detection detection of Salmonella is 104cfu/ml
The sensitivity of Figure 11 detection C.perfringens is 103cfu/ml
The sensitivity of Figure 12 detection Bacillus cercuses is 103cfu/ml
The sensitivity of Figure 13 detection campylobacter jejunis is 10cfu/ml
Specific embodiment
The present invention is described in detail with reference to specific embodiment, can be better understood from the present invention.However, embodiment is retouched The content stated is merely to illustrate the present invention, rather than in order to limit the scope of the present invention and application.
Embodiment 1：
Detection 13 kinds of primers and probe of diarrhoea pathogenic bacteria, are synthesized, in reality by Invitrogen (Shanghai) Trading Co., Ltd. Border using when three kinds of bacteriums primer pair and probe be divided into one group, it is specific as follows：
First group of amplification Staphylococcus aureus (Staphylococcus aureus) nuc genes, shigella dysenteriae (Shigella) IpaH genes, vibrio parahaemolytious (Vibrio Parahemolyticus) tlh gene primers pair, including following three pairs of primers and spy Pin：
Detect the primer pair and probe of staphylococcus aureus nuc genes：
F1(SEQ ID NO.：1)：ATCCTAAAAAAGGTGTAGAG,
R1(SEQ ID NO.：2)：TATCAGTTCTTTGACCTTTG,
P1(SEQ ID NO.：3)：FAM-ATATGGTCCTGAAGCAAGTGCA-MGB；
Detect the primer and probe of Shigella Gene ipaH Using：
F2(SEQ ID NO.：4)：ATAAAGTCAGAACTCTCC,
R2(SEQ ID NO.：5)：AACGCATTTCCTTCACGG,
P2(SEQ ID NO.：6)：VIC-ATGAGATAGAAGTCTACCTGGCCTTCC-TAMRA；
Detect the primer and probe of tlh gene of vibrio parahaemolyticus：
F3SEQ ID NO.：7：TCAACCGCTCATCGTCTGT,
R3SEQ ID NO.：8：CTGCGACATAGCGGTGAGT,
P3SEQ ID NO.：9：CY5-CACCCACGCATTGCGCTCTGAGTGT-BQ2；
Second group of amplification YE (Yersinia enterocolitica) ail genes, clostridium difficile (Clostridium difficile) tpi genes, Plesiomonas shigelloides (Plesiomonas shigelloides) hugA bases The primer and probe of cause, including following three pairs of primers and probe：
Detect the primer and probe of YE ail genes：
F4SEQ ID NO.：10：TAATAGGTTCGTTTGCTTATACC,
R4SEQ ID NO.：11：GCGGAAAGATGGCCCCATTGTTA,
P4SEQ ID NO.：12：FAM-CGATTTCTTCTATGGCAGTAATAAGT–TAMRA；
Detect the primer and probe of clostridium difficile tpi genes：
F5SEQ ID NO.：13:ACTGATGAAACTGTAAACA,
R5SEQ ID NO.：14:AACATCTTTAGTTTTTCCA,
P5SEQ ID NO.：15:HEX-CCCAATATTATGTGTTGGAGAAACTTTA-TAMRA；
Detection Plesiomonas shigelloides hugA genes：
F6SEQ ID NO.：16：TGACTGCTTTGAGCGCGCC,
R6SEQ ID NO.：17：AATCACAAGATGAATTGCC,
P6SEQ ID NO.：18：CY5-AGTGGTGTAGAGATCCACCAAGGTGTAG-BQ2；
3rd group of amplification Listeria (Listeria monocytogenes) hlyA genes, EHEC O157:H7 (E.coli O157:H7) rfbE genes, the primer of vibrio parahaemolytious (Vibrio Parahemolyticus) tlh genes and spy Pin, including following three pairs of primers and probe：
Expand the primer and probe of Listeria hlyA genes：
F7SEQ ID NO.：19：CAGGTGATGTAGAACTGACAAA,
R7SEQ ID NO.：20：GCACCTTTTTTCAAAATATCTC,
P7SEQ ID NO.：21:FAM-AAAGCCGTAATTTACGGTGGCTCCG-TAMRA；
Amplification EHEC O157:H7rfbE gene primers and probe：
F8SEQ ID NO.：22:TTTCGTTGATTCAGATAATG,
R8SEQ ID NO.：23:AAATTTCTACTTTTGGCCAG,
P8SEQ ID NO.：24:HEX-TAGTGACATAGAACAAAAAATCACTAA-TAMRA；
Amplification tlh gene of vibrio parahaemolyticus primer and probe：
F3SEQ ID NO.：7：TCAACCGCTCATCGTCTGT,
R3SEQ ID NO.：8:CTGCGACATAGCGGTGAGT,
P3SEQ ID NO.：9:CY5-CACCCACGCATTGCGCTCTGAGTGT-BQ2；
4th group of amplification Aeromonas (Aeromonas) gcat gene, detection of Salmonella (salmonella spp) invA genes, C.perfringens (C.perfringens) cpa gene primers pair and probe, including following three pairs of primers and probe：
Expand the primer and probe of Aeromonas gcat genes：
F9SEQ ID NO.：25：AAGGCGGTGCCACTGCCGT,
R9SEQ ID NO.：26：TCCGGCTTGAAGCTGTCTT,
P9SEQ ID NO.：27：FAM-TATCAGGTCATCAACAACCTGGACT-TAMRA；
Expand the primer and probe of detection of Salmonella invA genes：
F10SEQ ID NO.：28：TATTGGCGATAGCCTGGCGG,
R10SEQ ID NO.：29：TCGGCATCAATACTCATCTG,
P10SEQ ID NO.：30：Vic-AGTTTATCGTTATTACCAAAGGT-MGB；
Expand the primer and probe of C.perfringens cpa genes：
F11SEQ ID NO.：31:AGGGAAATCAATATACTATA,
R11SEQ ID NO.：32:TGAGAGTTAGCTAGAGTTAC,
P11SEQ ID NO.：33:CY5-AGTCATGCTAGCATGAGTCATAGTT-BQ2；
5th group of amplification staphylococcus aureus nuc genes (Staphylococcus aureus), Bacillus cercus The primer and probe of (Bacillus cereus) cesB genes, campylobacter jejuni mapA (Campylobacter jejuni), bag Include following three pairs of primers and probe：
Expand the primer and probe of staphylococcus aureus nuc genes：
F1SEQ ID NO.：1:ATCCTAAAAAAGGTGTAGAG,
R1SEQ ID NO.：2:TATCAGTTCTTTGACCTTTG,
P1SEQ ID NO.：3:FAM-ATATGGTCCTGAAGCAAGTGCA-MGB；
Expand the primer and probe of Bacillus cercus cesB genes：
F12SEQ ID NO.：34:CTGATGGGTTTAATGTAATGAA,
R12SEQ ID NO.：35:GTCTGACGTAATTCTGCTAATA,
P12SEQ ID NO.：36:VIC-ATTATAGTCGGAGTAGCAAGGGATAA–MGB；
The primer and probe of amplification campylobacter jejuni mapA：
F13SEQ ID NO.：37:TGCTCCATAAGATATAAATT,
R13SEQ ID NO.：38:TTTATACTTATTTAAAACAA,
P13SEQ ID NO.：39:CY5-CATAAGGTGAATTTTGATCGTTATTGT-BQ2.
Embodiment 2：The preparation method of kit.
(1) PCR reaction solutions：10*PCR reaction buffers, Q-solution, archaeal dna polymerase, Mg2+ and dNTPs, -20 DEG C Preserve；
(2) primer and probe mixed liquor：By SEQ ID NO.：Nucleotide sequence shown in 1~39 transfer to Invitrogen (on Sea) trade Co., Ltd's synthesis, then according to packet, three kinds of primer pairs and probe are mixed in a pipe, are dissolved with distilled water, often The final concentration of one primer is 1 μm of ol/L, -20 DEG C of preservations；
(3) positive control：Contain 13 kinds of diarrhoea pathogenic bacterial genomes DNA respectively, wherein, each contains drug resistant gene Bacterial genomes DNA concentration is 10ng/ μ L, -20 DEG C of preservations；
(4) negative control：Genome of E.coli DNA concentration is 100ng/ μ L, -20 DEG C of preservations.
Embodiment 3：Detection method.
Instrument：Roche480 fluorescent quantitative PCR detectors, BECKMAN22R is desk-top Micro refrigerated centrifuge, Eppendorf 5810R tabletop refrigerated centrifuges, granary Hua Lida laboratory equipments company WH-866 types Turbula shaker.
(1) fecal sample pre-treatment：1. sample pre-treatments：Weigh the aseptic PBS of 1g fecal specimens 9ml to suspend, acutely shake After swinging 15min, 200r/min is centrifuged 3 times, each 5min, collects supernatant；Then 5000r/min is centrifuged 3min, and collects thalline is sunk Form sediment, suspended with 1ml PBS and precipitated, repeated centrifugation, untill supernatant is substantially limpid；Last suspension with 1ml PBS again is collected into Precipitation, put in 2ml Eppendorf pipes -20 and save backup.
(2) preparation of bacterial genomes DNA profiling：With reference to disclosed document, different types of bacterium sample, using corresponding Commercialization genome DNA extraction kit, according to kit specification prepare bacterial genomes DNA, as PCR reaction templates It is standby.
(3) the bacterial genomes DNA for being obtained with step (1) is carried out carefully as template using 13 pairs of specific primers and probe Bacterium gene magnification detection, specifically includes following steps；
(3a) PCR reaction solutions are prepared：Each component from -20 DEG C of refrigerator taking-up embodiments 2, room temperature is melted, is put on ice chest It is standby.Within sample-adding is first 10 minutes, by detection sample number configuration PCR reaction solution X μ l：
(the μ l distilled waters of 23.5+7.5 μ l primer mixed liquors of μ l PCR reaction solutions+14) × (+1 part of positive of n parts of sample is right for X= According to+1 part of blank of+1 part of negative control).
After vibration is mixed, 2000rpm centrifugation 5s are dispensed into 96 hole PCR reaction plates by the μ l of every hole 45, go to sample preparation Area is standby.
(3b) is loaded：To in the reacting hole for having dispensed reagent, being separately added into the μ l of tested bacteria sample DNA template 5 (if sample Pyrolysis product is stored in -20 DEG C, using preposition thaw at RT, 5min is centrifuged with 13000rpm), Positive control wells add 5 μ l sun Property control, negative control hole add 5 μ l negative controls, blank control wells add 5 μ l distilled waters, be loaded range request and grasped on ice Make.Sealed membrane is posted, 3000rpm centrifugation 30s are then placed in PCR instrument.
(3c) PCR is expanded：Roche480 quantitative real time PCR Instruments carry out bacterial aminoglycoside class resistance base Because of detection, reaction condition is as follows：94 DEG C of predegeneration 5min；94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 20s, 45 circulations, fluorescent collecting O'clock at 58 DEG C, detection pattern is set to sonde method, and reaction volume is set to 50.Preserve file, operation.
(3d) interpretation of result, specifically includes following steps：
Roche480 fluorescent PCR detectors click on analysis, choose quantitative model, into analysis window, Selected noise line, threshold value is set as (can suitably being adjusted according to actual conditions just above the peak of random noise line It is whole), and positive curve is without flex point, it is ensured that the numerical value under noise line is consistent with the thresholding under analysis item.Blank pair is tested every time It is as a result qualified according to hole without Ct values.Click on and calculate, be calculated automatically from the CT values of each test.
(3e) yin and yang attribute reference substance result standard and treatment, specifically judge in accordance with the following steps：
The Ct values of negative controls PCR amplifications should be without numerical value or more than 40 and growth curve is irregular；Positive reference substance PCR Expand the S-type curve of growth curve and CT values are less than 37.
(4) the sample drug resistant gene testing result judgement obtained by step (3), specifically according to following standard：
(4a), if the not S-type curve of growth curve or Ct values are without numerical value or more than 40, it is feminine gender to sentence sample results.
(4b) is if the S-type curve of growth curve and Ct values<40, then judge by the following method：
If the CT of sample<37, then it is the positive to sentence sample results；
Detection sample Ct >=37, prepare PCR reaction solutions and enter performing PCR amplification again, if review result growth curve is not in S Type curve or Ct values are that without numerical value or more than 40, then it is feminine gender to sentence sample results.If growth curve is S-type and Ct values are still small It is the positive that sample results are sentenced in 40.
Embodiment 4：The sensitivity analysis of diarrhoea pathogenic bacteria detection kit
Reference culture is tuned into 1 × 106To 1 × 10cfu/ml bacterium solutions, then extracting DNA carries out sensitivity technique.
The detection of visible 13 diarrhoea pathogenic bacterium is limited to 10 in accompanying drawing 1~134~10cfu/ml.
SEQUENCE LISTING
<110>Hangzhou Dean Hangzhou D.A. Diagnostics Center Co., Ltd. of Bioisystech Co., Ltd
<120>Primer and its application of various diarrhoea pathogenic bacterias are detected simultaneously
<130> SG20170223001
<160> 39
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>F1 primers
<400> 1
atcctaaaaa aggtgtagag 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>R1 primers
<400> 2
tatcagttct ttgacctttg 20
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223>P1 probes
<400> 3
atatggtcct gaagcaagtg ca 22
<210> 4
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>F2 primers
<400> 4
ataaagtcag aactctcc 18
<210> 5
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>R2 primers
<400> 5
aacgcatttc cttcacgg 18
<210> 6
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223>P2 probes
<400> 6
atgagataga agtctacctg gccttcc 27
<210> 7
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>F3 primers
<400> 7
tcaaccgctc atcgtctgt 19
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>R3 primers
<400> 8
ctgcgacata gcggtgagt 19
<210> 9
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223>P3 probes
<400> 9
cacccacgca ttgcgctctg agtgt 25
<210> 10
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223>F4 primers
<400> 10
taataggttc gtttgcttat acc 23
<210> 11
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223>R4 primers
<400> 11
gcggaaagat ggccccattg tta 23
<210> 12
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223>P4 probes
<400> 12
cgatttcttc tatggcagta ataagt 26
<210> 13
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>F5 primers
<400> 13
actgatgaaa ctgtaaaca 19
<210> 14
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>R5 primers
<400> 14
aacatcttta gtttttcca 19
<210> 15
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223>P5 probes
<400> 15
cccaatatta tgtgttggag aaacttta 28
<210> 16
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>F6 primers
<400> 16
tgactgcttt gagcgcgcc 19
<210> 17
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>R6 primers
<400> 17
aatcacaaga tgaattgcc 19
<210> 18
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223>P6 probes
<400> 18
agtggtgtag agatccacca aggtgtag 28
<210> 19
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223>F7 primers
<400> 19
caggtgatgt agaactgaca aa 22
<210> 20
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> R7
<400> 20
gcaccttttt tcaaaatatc tc 22
<210> 21
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223>P7 probes
<400> 21
aaagccgtaa tttacggtgg ctccg 25
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>F8 primers
<400> 22
tttcgttgat tcagataatg 20
<210> 23
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>R8 primers
<400> 23
aaatttctac ttttggccag 20
<210> 24
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223>P8 probes
<400> 24
tagtgacata gaacaaaaaa tcactaa 27
<210> 25
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>F9 primers
<400> 25
aaggcggtgc cactgccgt 19
<210> 26
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>R9 primers
<400> 26
tccggcttga agctgtctt 19
<210> 27
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223>P9 probes
<400> 27
tatcaggtca tcaacaacct ggact 25
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>F10 primers
<400> 28
tattggcgat agcctggcgg 20
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>R10 primers
<400> 29
tcggcatcaa tactcatctg 20
<210> 30
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223>P10 probes
<400> 30
agtttatcgt tattaccaaa ggt 23
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>F11 primers
<400> 31
agggaaatca atatactata 20
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>R11 primers
<400> 32
tgagagttag ctagagttac 20
<210> 33
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223>P11 probes
<400> 33
agtcatgcta gcatgagtca tagtt 25
<210> 34
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223>F12 primers
<400> 34
ctgatgggtt taatgtaatg aa 22
<210> 35
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223>R12 primers
<400> 35
gtctgacgta attctgctaa ta 22
<210> 36
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223>P12 probes
<400> 36
attatagtcg gagtagcaag ggataa 26
<210> 37
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>F13 primers
<400> 37
tgctccataa gatataaatt 20
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>R13 primers
<400> 38
tttatactta tttaaaacaa 20
<210> 39
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223>P13 probes
<400> 39
cataaggtga attttgatcg ttattgt 27

Claims (5)

1. the primer of various diarrhoea pathogenic bacterias is detected simultaneously, it is characterised in that including following five groups of primers：
First group of primer：Including following three pairs of primers and probe：
Pair of primers and probe：
F1 SEQ ID NO.：1：ATCCTAAAAAAGGTGTAGAG,
R1 SEQ ID NO.：2：TATCAGTTCTTTGACCTTTG,
P1 SEQ ID NO.：3：FAM-ATATGGTCCTGAAGCAAGTGCA-MGB；
Second pair of primer and probe：
F2 SEQ ID NO.：4：ATAAAGTCAGAACTCTCC,
R2 SEQ ID NO.：5：AACGCATTTCCTTCACGG,
P2 SEQ ID NO.：6：VIC-ATGAGATAGAAGTCTACCTGGCCTTCC-TAMRA；
3rd pair of primer and probe：
F3 SEQ ID NO.：7：TCAACCGCTCATCGTCTGT,
R3 SEQ ID NO.：8：CTGCGACATAGCGGTGAGT,
P3 SEQ ID NO.：9：CY5-CACCCACGCATTGCGCTCTGAGTGT-BQ2；
Second group of primer：Including following three pairs of primers and probe：
Pair of primers and probe：
F4 SEQ ID NO.：10:TAATAGGTTCGTTTGCTTATACC,
R4 SEQ ID NO.：11:GCGGAAAGATGGCCCCATTGTTA,
P4 SEQ ID NO.：12：FAM-CGATTTCTTCTATGGCAGTAATAAGT–TAMRA；
Second pair of primer and probe：
F5 SEQ ID NO.：13:ACTGATGAAACTGTAAACA,
R5 SEQ ID NO.：14:AACATCTTTAGTTTTTCCA,
P5 SEQ ID NO.：15:HEX-CCCAATATTATGTGTTGGAGAAACTTTA-TAMRA；
3rd pair of primer and probe：
F6 SEQ ID NO.：16：TGACTGCTTTGAGCGCGCC,
R6 SEQ ID NO.：17：AATCACAAGATGAATTGCC,
P6 SEQ ID NO.：18：CY5-AGTGGTGTAGAGATCCACCAAGGTGTAG-BQ2；
3rd group of primer：Including following three pairs of primers and probe：
Pair of primers and probe：
F7 SEQ ID NO.：19：CAGGTGATGTAGAACTGACAAA,
R7 SEQ ID NO.：20：GCACCTTTTTTCAAAATATCTC,
P7 SEQ ID NO.：21:FAM-AAAGCCGTAATTTACGGTGGCTCCG-TAMRA；
Second pair of primer and probe：
F8 SEQ ID NO.：22:TTTCGTTGATTCAGATAATG,
R8 SEQ ID NO.：23:AAATTTCTACTTTTGGCCAG,
P8 SEQ ID NO.：24:HEX-TAGTGACATAGAACAAAAAATCACTAA-TAMRA；
3rd pair of primer and probe：
F3 SEQ ID NO.：7：TCAACCGCTCATCGTCTGT,
R3 SEQ ID NO.：8:CTGCGACATAGCGGTGAGT,
P3 SEQ ID NO.：9:CY5-CACCCACGCATTGCGCTCTGAGTGT-BQ2；
4th group of primer：Including following three pairs of primers and probe：
Pair of primers and probe：
F9 SEQ ID NO.：25：AAGGCGGTGCCACTGCCGT,
R9 SEQ ID NO.：26：TCCGGCTTGAAGCTGTCTT,
P9 SEQ ID NO.：27：FAM-TATCAGGTCATCAACAACCTGGACT-TAMRA；
Second pair of primer and probe：
F10 SEQ ID NO.：28：TATTGGCGATAGCCTGGCGG,
R10 SEQ ID NO.：29：TCGGCATCAATACTCATCTG,
P10 SEQ ID NO.：30：Vic-AGTTTATCGTTATTACCAAAGGT-MGB；
3rd pair of primer and probe：
F11 SEQ ID NO.：31:AGGGAAATCAATATACTATA,
R11 SEQ ID NO.：32:TGAGAGTTAGCTAGAGTTAC,
P11 SEQ ID NO.：33:CY5-AGTCATGCTAGCATGAGTCATAGTT-BQ2；
5th group of primer：Including following three pairs of primers and probe：
Pair of primers and probe：
F1 SEQ ID NO.：1:ATCCTAAAAAAGGTGTAGAG,
R1 SEQ ID NO.：2:TATCAGTTCTTTGACCTTTG,
P1 SEQ ID NO.：3:FAM-ATATGGTCCTGAAGCAAGTGCA-MGB；
Second pair of primer and probe：
F12 SEQ ID NO.：34:CTGATGGGTTTAATGTAATGAA,
R12 SEQ ID NO.：35:GTCTGACGTAATTCTGCTAATA,
P12 SEQ ID NO.：36:VIC-ATTATAGTCGGAGTAGCAAGGGATAA–MGB；
3rd pair of primer and probe：
F13 SEQ ID NO.：37:TGCTCCATAAGATATAAATT,
R13 SEQ ID NO.：38:TTTATACTTATTTAAAACAA,
P13 SEQ ID NO.：39:CY5-CATAAGGTGAATTTTGATCGTTATTGT-BQ2.
2. according to claim 1 while detecting the primer of various diarrhoea pathogenic bacterias, it is characterised in that described diarrhoeal diseases Opportunistic pathogen includes：Staphylococcus aureus, shigella dysenteriae, vibrio parahaemolytious, YE, Clostridium difficile bacillus, neighbour Monad, Listeria, EHEC O157：H7, Aeromonas, detection of Salmonella, C.perfringens, Bacillus cercus and Campylobacter jejuni.
3. the kit of various diarrhoea pathogenic bacterias is detected simultaneously, it is characterised in that including following reagent：
(1) PCR reaction solutions：10*PCR buffer solutions, Q buffer solutions, archaeal dna polymerase, Mg2+And dNTPs, the Q bufferings include as follows Component：Glycine betaine 2.7M, DMSO6.7%, DTT6.7mM, BSA0.055mg/ml；
(2) primer and probe mixed liquor:By SEQ ID NO.：Nucleotide sequence shown in 1~39 is grouped according to claim 1, Final concentration of 1 μm of ol/L of every kind of primer；
(3) positive control：Containing diarrhoea pathogenic bacterial genomes DNA, wherein, DNA is dense for each diarrhoea pathogenic bacterial genomes It is 10ng/ μ L to spend；
(4) negative control：Genome of E.coli DNA, concentration is 100ng/ μ L.
4. application of the kit of various diarrhoea pathogenic bacterias in diarrhoea pathogenic bacteria is detected is detected simultaneously described in claim 3.
5. application according to claim 1, it is characterised in that the diarrhoea pathogenic bacteria includes：Staphylococcus aureus, will Congratulate bacterium, vibrio parahaemolytious, YE, Clostridium difficile bacillus, Plesiomonas, Listeria, EHEC O157：Application in H7, Aeromonas, detection of Salmonella, C.perfringens, Bacillus cercus and campylobacter jejuni.
CN201710167845.3A 2017-03-21 2017-03-21 Primer and its application of various diarrhoea pathogenic bacterias are detected simultaneously Pending CN106868160A (en)

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CN110878366A (en) * 2019-11-27 2020-03-13 安序源生物科技(深圳)有限公司 Nucleic acid composition, detection kit for intestinal pathogenic bacteria and use method of detection kit
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Application publication date: 20170620