CN105695563A - Primer sequence used in escherichia coli and staphylococcus aureus 23S-5S rRNA gene sequencing identification method - Google Patents
Primer sequence used in escherichia coli and staphylococcus aureus 23S-5S rRNA gene sequencing identification method Download PDFInfo
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- CN105695563A CN105695563A CN201510214122.5A CN201510214122A CN105695563A CN 105695563 A CN105695563 A CN 105695563A CN 201510214122 A CN201510214122 A CN 201510214122A CN 105695563 A CN105695563 A CN 105695563A
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Abstract
The invention relates to a primer sequence used in an escherichia coli and staphylococcus aureus 23S-5S rRNA gene sequencing identification method, and aims to provide the primer sequence used in the 23S-5S rRNA gene sequencing identification method for identification of escherichia coli and staphylococcus aureus pathogenic bacteria. The primer sequence used in the escherichia coli and staphylococcus aureus 23S-5S rRNA gene sequencing identification method is provided. With use of the sequence, a part of characteristic gene fragments in 23S-5S rRNA can be determined, and the sequence is used for accurately identify the escherichia coli and the staphylococcus aureus. Primers provided by the invention can be used for accurately sequencing the 23S-5S rRNA gene of the escherichia coli and the staphylococcus aureus; according to results of sequencing, and with combination of free public gene database retrieval, the two pathogenic bacteria can be accurately identified.
Description
Technical field
The present invention relates to a kind of primer sequence for escherichia coli and staphylococcus aureus 23S-5SrRNA gene sequencing identification method。
Background technology
The authentication method of pathogen is varied, and the Instrumental Analysis based on biochemical reaction that remains of current relatively broad application is identified, but very not accurate in some cases。Rise along with Protocols in Molecular Biology so that the method for discriminating bacteria is more deep with accurately on a molecular scale, and order-checking rule is one of them。Prokaryote rRNA gene 5 ' end is followed successively by 3 ' ends: 5 '-16S (1540bp)-ITS1-23S (2900bp)-ITS2 (70bp)-5S (120bp)-3 '。Wherein, 16SrRNA contains more region of variability and conserved region because of it, and molecular size is suitable for, the Main Basis that its sequence analysis is classified as microflora is widely used, technology is comparatively ripe, data base is comparatively perfect, but still scarce for the differentiation dynamics between the interior strain of close kind and kind。16S-23SrRNA because of its faster tempo of evolution be applied to the 16SrRNA strain in close relations that cannot differentiate or plant in discriminating between strain, be that the former supplements very well。Even so, some antibacterial such as staphylococcus aureus, escherichia coli etc., between its 16S-23SrDNA, district is more complicated, and size between different copies, gene comparision are close, and sequencing result is not easily explained。23SrRNA molecule is big, only has the sequence of minority bacterium to be in the news so far, and data base is imperfection very, and in different bacterium kind, the variability of this fragment is different, therefore in division bacteria with fail extensive use in identifying。5SrRNA was once used in environment the qualification of microorganism, but because fragment is little, and it is little to carry hereditary information amount, equally, is not also widely adopted in microbiological analysis is identified。23S-5S sequence of intervals is shorter due to sequence, so far, both at home and abroad to 23S-5S sequence of intervals identifying that the report of the research in microorganism is very few, is close to blank。This research each designs primer for the 23S back segment-ITS2-5S sequence of two kinds of bacterium, establish method Fluorescence PCR assay and product direct Sequencing combined, obtain they 23S back segment-ITS2-5S fragments and carry out sequence analysis, sequence is submitted to NCBI website again carry out nucleotideblast and compare, obtains the highest coupling strain of similarity to determine its genotyping result。It is demonstrated experimentally that 23S-5S sequence is used for identifying staphylococcus aureus, escherichia coli accurately and reliably as genetic marker。
Summary of the invention
It is an object of the invention to open one and can be used for escherichia coli and staphylococcus aureus cause of disease
The primer sequence of the 23S-5SrRNA gene sequencing identification method of the dientification of bacteria。
A kind of primer sequence for escherichia coli and staphylococcus aureus 23S-5SrRNA gene sequencing identification method。Use this sequence can measure Partial Feature genetic fragment in 23S-5SrRNA, for precise Identification escherichia coli and staphylococcus aureus。
Following table is table 1, for the primer sequence for escherichia coli and staphylococcus aureus 23S-5SrRNA gene sequencing identification method
Based on above-mentioned purpose, the present invention adopts embodiment in detail below:
Adopting FTA card nucleic acid preservation pathogen nucleic acid, real-time fluorescence PCR technology amplification target fragment, upper machine carries out dideoxy sequencing。
The fluorescent PCR quick sequencing reagent suit developed based on the present invention includes following reagent:
1) 1, FTA card。
2) PCR reactant liquor 1 is managed, built-in SYBRGREEN Fluorescence PCR liquid, including Taq enzyme, dNTP, containing Mg2+Buffer, SYBRGREEN solution。
3) each 1 pipe of upstream and downstream primer, built-in target sequence amplification and sequencing primer。
4) dideoxy sequencing reagent is a set of, including BigDyeTerminatorv3.1SequencingBuffer (5 ×), BigDyeMix。
5) order-checking product purification reagent is a set of, including each 1 pipe of XTerminatorsolution, SAMsolution。
6) deionized water 1 is managed, the built-in sterilizing deionized water without DNA enzymatic。
Based on the quick sequence measurement based on fluorescent PCR that the present invention sets up, follow these steps to carry out:
1) preparation of FTA sample card
Bacterial suspension is after suitably dilution (about 6 × 104To 6 × 107CFU/mL), drawing 100ul and drip on FTA card, room temperature is dried standby。
2) SYBRGreen real-time fluorescence PCR directly expands target fragment
Preparation reaction system: preparation SYBRGreen real-time fluorescence PCR reaction system routinely, the final concentration adding upstream and downstream primer is 100ng/uL, and the scraps of paper getting diameter 0.5mm at FTA card with card punch add system, and final volume is 20uL。Fluorescent PCR directly expands: using quantitative real time PCR Instrument to expand, amplification reaction condition is, 95 DEG C of 2min, [95 DEG C of 10s, 51 DEG C of 20S, 72 DEG C of 40S (fluorescent collecting)] 35 circulations, 72 DEG C of 10min。Pcr amplification effect assessment: the standard that fluorescence PCR products is qualified be Cp value less than 30 and melting curve display the obvious main peak of only one of which, be otherwise amplified production defective。
3) order-checking PCR reaction
If fluorescent PCR amplified production is qualified, PCR primer is diluted 5-10 times according to Cp value size, take the product 1ul diluted and be placed in PCR pipe, add 4ulBigDyeTerminatorv3.1SequencingBuffer (5 ×) again, the final concentration of 100ng/uL of sequencing primer (upstream or downstream primer), final volume is 10uL, uses grads PCR instrument to expand。Amplification condition is: 98 DEG C of 2min, (96 DEG C of 10S, 50 DEG C of 5s, 60 DEG C of 4min) 25 circulations。
4) product purification, capillary electrophoresis and sequence read, analyze and comparison
96 orifice plates add product 1uL, SAMsolution9uL, BigDyeXTerminatorsolution2uL。The centrifugal 2min of concussion 5min, 2000g, covers the capping of order-checking silica gel, carries out capillary electrophoresis with Genetic Analyser, and the complete machine of electrophoresis reads automatically according to order-checking spectrogram line order row of going forward side by side, and can obtain target fragment gene order。Sequencing result uses " nucleotideblast " function on NCBI website to carry out sequence alignment, data base selects " Nucleotidecollection (nr/nt) ", chooses comparison result matching rate the highest (being generally first term) as final genotyping result。
Present invention have the advantage that。
1. the 23S-5SrRNA gene of escherichia coli and staphylococcus aureus can correctly be checked order by primer provided by the invention, according to this sequencing result, in conjunction with free public gene data library searching, can identify both pathogen exactly。
2. the 23S-5SrRNA gene sequencing identification method of escherichia coli provided by the invention and staphylococcus aureus, can make up some deficiency of general 23S-5SrRNA gene sequencing qualification result, increase accuracy and the judgement index of Bacteria Identification。
3. the invention of the 23S-5SrRNA gene sequencing identification method of escherichia coli and staphylococcus aureus so that promote gene sequencing method at laboratories and identify that pathogenic bacterium are possibly realized。
Accompanying drawing explanation
Fig. 1 is the escherichia coli and the staphylococcus aureus 23S-5SrRNA gene real-time fluorescent PCR amplification design sketch that utilize reagent and the method that the present invention relates to, and two kinds of bacterium 23S-5SrRNA gene real-time fluorescent PCR amplification design sketchs, A is amplification curve diagram；B is melting curve figure。
Fig. 2 is the typical 23S-5SrRNA gene sequencing spectrogram of escherichia coli and the staphylococcus aureus utilizing reagent and the method that the present invention relates to；Typical case's 23S-5SrRNA gene sequencing spectrogram。
Embodiment: treat order-checking pathogen identification 8 strain: from clinical samples isolated strains, grow lawn for inclined-plane。Passing through traditional biochemical reaction to identify, wherein 4 strains are escherichia coli, and 4 strains are staphylococcus aureus。
Embodiment。
Treat order-checking pathogen identification 8 strain: from clinical samples isolated strains, grow lawn for inclined-plane。Passing through traditional biochemical reaction to identify, wherein 4 strains are escherichia coli, and 4 strains are staphylococcus aureus。
Detecting step: application mentioned reagent and method detect。
1) extraction of the process of sample and DNA profiling
Above-mentioned isolated strains bacterial suspension is after suitably dilution (about 6 × 104To 6 × 107CFU/mL), drawing 100ul and drip on FTA card, room temperature is dried standby。
2) SYBRGreen real-time fluorescence PCR reaction system preparation
After being taken out from refrigerator by SYBRGreen real-time fluorescence PCR, putting room temperature and melt, 2000rpm is centrifuged 10sec。According to the form below calculates reagent dosage。
Table 2SYBRGreen real-time fluorescence PCR reaction system preparation table
| Reagent name | PCR reactant liquor. | Upstream and downstream primer | Sterilizing deionized water |
| Consumption μ L | 10×8.5 | Each 1 × 8.5 | 8×8.5 |
By 85 μ LPCR reactant liquors, 8.5 μ L forward primer, 8.5 μ L downstream primers, 68 μ L sterilizing deionized waters, it is separately added in 1.5mL centrifuge tube, after mixing, 2000rpm is centrifuged 10sec, adds 20 μ L/ holes respectively in the positive reaction hole set, negative reaction hole and example reaction hole。
3) application of sample
Get 1 addition system of the scraps of paper of diameter 0.5mm with card punch at FTA card, seal reacting hole with reaction film, be placed on fluorescent PCR instrument and detect。
4) response parameter and pcr amplification effect assessment
Fluorescent PCR instrument selects FAM passage, and amplification reaction condition is, 95 DEG C of 2min, [95 DEG C of 10s, 51 DEG C of 20S, 72 DEG C of 40S (fluorescent collecting)] 35 circulations, 72 DEG C of 10min。Pcr amplification effect assessment: Cp(or Ct) value less than 30 and melting curve show an obvious main peak, be otherwise amplified production defective。
5) order-checking PCR reaction
If fluorescent PCR amplified production is qualified, PCR primer is diluted 5-10 times according to Cp value size, take the product 1ul diluted and be placed in PCR pipe, add 4ulBigDyeTerminatorv3.1SequencingBuffer (5 ×) again, the final concentration of 100ng/uL of sequencing primer (upstream or downstream primer), final volume is 10uL, uses grads PCR instrument to expand。Amplification condition is: 98 DEG C of 2min, (96 DEG C of 10S, 50 DEG C of 5s, 60 DEG C of 4min) 25 circulations。
6) product purification, capillary electrophoresis, sequence read, analyze and comparison
96 orifice plates add product 1uL, SAMsolution9uL, BigDyeXTerminatorsolution2uL。The centrifugal 2min of concussion 5min, 2000g, covers the capping of order-checking silica gel, carries out capillary electrophoresis with Genetic Analyser, and the complete machine of electrophoresis reads automatically according to order-checking spectrogram line order row of going forward side by side, and can obtain target fragment gene order。Sequencing result uses " nucleotideblast " function on NCBI website to carry out sequence alignment, data base selects " Nucleotidecollection (nr/nt) ", chooses comparison result matching rate the highest (being generally first term) as final genotyping result。
Following table is staphylococcus aureus and colon bacillus sequence analysis and comparison result thereof
Claims (2)
1. the primer sequence for escherichia coli and staphylococcus aureus 23S-5SrRNA gene sequencing identification method, it is characterised in that described primer sequence includes:
。
2. one kind is used for escherichia coli and staphylococcus aureus 23S-5SrRNA gene sequencing identification method, adopt FTA card nucleic acid preservation pathogen nucleic acid, real-time fluorescence PCR technology amplification target fragment, upper machine carries out dideoxy sequencing, and the fluorescent PCR quick sequencing reagent suit developed based on the present invention includes following reagent:
1) 1, FTA card;
2) PCR reactant liquor 1 is managed, built-in SYBRGREEN Fluorescence PCR liquid, including Taq enzyme, dNTP, containing Mg2+Buffer, SYBRGREEN solution;
Each 1 pipe of upstream and downstream primer, built-in target sequence amplification and sequencing primer;
Dideoxy sequencing reagent is a set of, including BigDyeTerminatorv3.1SequencingBuffer (5 ×), BigDyeMix;
Order-checking product purification reagent is a set of, including each 1 pipe of XTerminatorsolution, SAMsolution;
Deionized water 1 is managed, the built-in sterilizing deionized water without DNA enzymatic;
Based on the quick sequence measurement based on fluorescent PCR that the present invention sets up, follow these steps to carry out:
1) preparation of FTA sample card
Bacterial suspension is after suitably dilution (about 6 × 104To 6 × 107CFU/mL), drawing 100ul and drip on FTA card, room temperature is dried standby；
2) SYBRGreen real-time fluorescence PCR directly expands target fragment
Preparation reaction system: preparation SYBRGreen real-time fluorescence PCR reaction system routinely, the final concentration adding upstream and downstream primer is 100ng/uL, and the scraps of paper getting diameter 0.5mm at FTA card with card punch add system, and final volume is 20uL；
Fluorescent PCR directly expands: using quantitative real time PCR Instrument to expand, amplification reaction condition is, 95 DEG C of 2min, [95 DEG C of 10s, 51 DEG C of 20S, 72 DEG C of 40S (fluorescent collecting)] 35 circulations, 72 DEG C of 10min；
Pcr amplification effect assessment: the standard that fluorescence PCR products is qualified be Cp value less than 30 and melting curve display the obvious main peak of only one of which, be otherwise amplified production defective；
3) order-checking PCR reaction
If fluorescent PCR amplified production is qualified, PCR primer is diluted 5-10 times according to Cp value size, take the product 1ul diluted and be placed in PCR pipe, add 4ulBigDyeTerminatorv3.1SequencingBuffer (5 ×) again, the final concentration of 100ng/uL of sequencing primer (upstream or downstream primer), final volume is 10uL, uses grads PCR instrument to expand；
Amplification condition is: 98 DEG C of 2min, (96 DEG C of 10S, 50 DEG C of 5s, 60 DEG C of 4min) 25 circulations；
4) product purification, capillary electrophoresis and sequence reading, analysis and comparison add product 1uL, SAMsolution9uL, BigDyeXTerminatorsolution2uL in 96 orifice plates；
The centrifugal 2min of concussion 5min, 2000g, covers the capping of order-checking silica gel, carries out capillary electrophoresis with Genetic Analyser, and the complete machine of electrophoresis reads automatically according to order-checking spectrogram line order row of going forward side by side, and can obtain target fragment gene order；
Sequencing result uses " nucleotideblast " function on NCBI website to carry out sequence alignment, data base selects " Nucleotidecollection (nr/nt) ", chooses comparison result matching rate the highest (being generally first term) as final genotyping result。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7002005B1 (en) * | 1997-07-21 | 2006-02-21 | Biotecon Diagnostics Gmbh | Analytical detection of Staphylococcus aureus |
| CN100473728C (en) * | 1999-09-24 | 2009-04-01 | 生物技术检测股份有限公司 | Nucleic acid molecules for detecting bacteria and phylogenetic units of bacteria |
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- 2015-04-30 CN CN201510214122.5A patent/CN105695563A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7002005B1 (en) * | 1997-07-21 | 2006-02-21 | Biotecon Diagnostics Gmbh | Analytical detection of Staphylococcus aureus |
| CN100473728C (en) * | 1999-09-24 | 2009-04-01 | 生物技术检测股份有限公司 | Nucleic acid molecules for detecting bacteria and phylogenetic units of bacteria |
Non-Patent Citations (1)
| Title |
|---|
| 陈霖祥等: "荧光PCR-16S rDNA测序法鉴定金黄色葡萄球菌的FTA卡制备DNA模板条件的优化", 《汕头大学医学院学报》 * |
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