CN104673791A - Specific DNA (deoxyribonucleic acid) sequence of NG (neisseria gonorrhoeae) - Google Patents
Specific DNA (deoxyribonucleic acid) sequence of NG (neisseria gonorrhoeae) Download PDFInfo
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- CN104673791A CN104673791A CN201510104122.XA CN201510104122A CN104673791A CN 104673791 A CN104673791 A CN 104673791A CN 201510104122 A CN201510104122 A CN 201510104122A CN 104673791 A CN104673791 A CN 104673791A
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- sequence
- specific dna
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- gonococcus
- deoxyribonucleic acid
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Abstract
The invention discloses a specific DNA (deoxyribonucleic acid) sequence of NG (neisseria gonorrhoeae). The sequence is obtained through PCR (polymerase chain reaction) amplification of a human genital tract NG infected specimen, and the sequence is determined to be a target gene sequence after blast comparison of the sequence. By means of the specific DNA sequence, the foundation for research of the action mechanism of NG molecules is laid, further, a method adopted by an established clinical detection technology has the advantages of rapidness, accuracy, simplicity and convenience in operation and the like, and the cost is lower.
Description
Technical field
The present invention relates to a kind of field of biomedicine technology, particularly relate to the specific DNA sequence of one section of gonococcus NG.
Background technology
Gonococcus NG is the pathogen that a kind of urinary system often infects, urethritis, trachelitis, salpingitis, microbemia, sacroiliitis etc. can be caused, the diseases such as spontaneous abortion, premature rupture of fetal membrane, premature labor and acute molten film amnionitis can be caused at pregnancy duration, diagnosis is the foundation of carrying out immediately and reliably treating exactly, can only diagnose at present according to clinical indication.PCR and gene chip diagnosis are one detection methods fast and accurately, and the diagnostic method of this class depends on the special nucleotide sequence of acquisition.
Summary of the invention
The invention reside in by information biology and Protocols in Molecular Biology, determine one section of gonococcus NG specific nucleotide sequence and PCR primer thereof, this sequence and corresponding PCR primer can be used for setting up the diagnosis that gene chip and PCR method carry out gonococcus disease.
Technical scheme of the present invention is: the present invention determines specific DNA sequence and the PCR primer thereof of one section of gonococcus NG, pcr amplification is carried out with primer, sequencing result is by manual check and correction, sequence assembly, and Blast similarity searching is carried out in NCBI, guarantee that gained sequence is target sequence, it is characterized in that, its sequence is as follows:
tcaccgtcta tatggaaacc gccgccgcac aaagcgacag cgataccgta cgcagcctgc 60
tgacgcgcga taaacggctc gacaacatcc gcttcatcgg caaggaagac ggtttggcgg 120
aattacagtc caacctcg 138
The sequence of primer:
Forward primer: tcaccgtctatatggaaa
Reverse primer: gaggttggactgtaattc
The sequence of probe:
accgtcttccttgccgatga
The specific DNA sequence of described gonococcus NG for diagnosing the probe sequence of gonococcal gene chip, and carries out pcr amplification by above-mentioned primer sequence, carries out hybridization check or be directly used in PCR diagnosis after mark.
The invention has the beneficial effects as follows: the acquisition of this sequence is not only the mechanism of action studying gonococcus quasi-molecule and lays the foundation, and the method that the clinical detection technique that the present invention sets up uses has the advantages such as quick, accurate, easy and simple to handle, and cost is also lower.
Embodiment
Below in conjunction with embodiment, the invention will be further described:
1, design of primers:
(1), sequence is searched: find relevant sequence by NCBI
(2), primed probe design: by primed probe design software, design at stronger one section of goal gene sequence-specific.
2, primer synthesis:
Primed probe all synthesizes in Sangon Biotech (Shanghai) Co., Ltd.;
3, extract
(1), the preparation of clinical samples DNA profiling:
Get a little patient urine or secretory product as sample, add appropriate physiological saline and be fully mixed, 2000r/min low-speed centrifugal 5min removes supernatant liquor, stays precipitation for subsequent use;
(2), sample DNA extracts:
A, get the sample doing pre-treatment a little and add 50ul lysate (1 × PCR Buffer, 0.45%NP-400.45%Tween-20,200ug/ml Proteinase K), sample is put people's 55 DEG C of water-baths 1 hour, 100 DEG C of water-bath 10min;
Under b, 12000r/rain condition, centrifugal 5min, gets supernatant for subsequent use;
4, increase
A, amplifing reagent: Anstart Taq Master Mix (luxuriant and rich with fragrance roc is biological, article No. A6120), the primed probe (concentration is all diluted to 10P) of NG, quantitative real time PCR Instrument (step one, ABI);
B, preparation of reagents: in table 1
Table 1 preparation of reagents table
C, amplification condition: in table 2
Table 2 amplification condition table
5, the clone identification of this gene
6, order-checking is entrusted
Choosing 3-5 the good pcr amplification product of effect send by check order after Sangon Biotech's purifying (order-checking the primer is identical with PCR the primer), what adopt is the order-checking of sanger method, the instrument of main employing is 3730XL sequenator, main agents is bigdye, is goal gene sequence after the calibrated splicing of gained gene order;
7, sequential analysis
Sequencing result by manual check and correction, sequence assembly, and carries out Blast similarity searching in NCBI, guarantees that gained sequence is target sequence.
The sequence of the primer of the present invention is:
Forward primer: tcaccgtctatatggaaa
Reverse primer: gaggttggactgtaattc
The sequence that the present invention records is:
tcaccgtcta tatggaaacc gccgccgcac aaagcgacag cgataccgta cgcagcctgc 60
tgacgcgcga taaacggctc gacaacatcc gcttcatcgg caaggaagac ggtttggcgg 120
aattacagtc caacctcg 138
Claims (2)
1. the specific DNA sequence of gonococcus NG, is characterized in that, its sequence is as follows:
tcaccgtcta tatggaaacc gccgccgcac aaagcgacag cgataccgta cgcagcctgc 60
tgacgcgcga taaacggctc gacaacatcc gcttcatcgg caaggaagac ggtttggcgg 120
aattacagtc caacctcg 138
The sequence of primer:
Forward primer: tcaccgtctatatggaaa
Reverse primer: gaggttggactgtaattc
The sequence of probe:
accgtcttccttgccgatga。
2. the application of the specific DNA sequence of gonococcus NG as claimed in claim 1, it is characterized in that, the specific DNA sequence of described gonococcus NG is for diagnosing the probe sequence of gonococcal gene chip, and carry out pcr amplification by above-mentioned primer sequence, carry out hybridization check after mark or be directly used in PCR diagnosis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510104122.XA CN104673791A (en) | 2015-03-10 | 2015-03-10 | Specific DNA (deoxyribonucleic acid) sequence of NG (neisseria gonorrhoeae) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510104122.XA CN104673791A (en) | 2015-03-10 | 2015-03-10 | Specific DNA (deoxyribonucleic acid) sequence of NG (neisseria gonorrhoeae) |
Publications (1)
| Publication Number | Publication Date |
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| CN104673791A true CN104673791A (en) | 2015-06-03 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510104122.XA Pending CN104673791A (en) | 2015-03-10 | 2015-03-10 | Specific DNA (deoxyribonucleic acid) sequence of NG (neisseria gonorrhoeae) |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1436852A (en) * | 2002-02-09 | 2003-08-20 | 昆明寰基生物芯片开发有限公司 | Specificity determination and application of one section of gonococcus DNA sequence |
| CN1465716A (en) * | 2002-06-26 | 2004-01-07 | 昆明寰基生物芯片开发有限公司 | Determination for specificity of one segmentum gonococcus DNA sequence (3) and use thereof |
| CN1465714A (en) * | 2002-06-26 | 2004-01-07 | 昆明寰基生物芯片开发有限公司 | One segmentum gonococcus DNA sequence (2) specificity detemination and use thereof |
| CN1465715A (en) * | 2002-06-26 | 2004-01-07 | 昆明寰基生物芯片开发有限公司 | Determination for specificity of one segmentum gonococcus DNA sequence (5) and use thereof |
| EP1777233A2 (en) * | 2001-02-12 | 2007-04-25 | Novartis Vaccines and Diagnostics S.r.l. | Gonococcal proteins and nucleic acids |
| CN103060453A (en) * | 2013-01-10 | 2013-04-24 | 湖南圣湘生物科技有限公司 | Kit for detecting neisseria gonorrheae (NG) |
-
2015
- 2015-03-10 CN CN201510104122.XA patent/CN104673791A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1777233A2 (en) * | 2001-02-12 | 2007-04-25 | Novartis Vaccines and Diagnostics S.r.l. | Gonococcal proteins and nucleic acids |
| CN1436852A (en) * | 2002-02-09 | 2003-08-20 | 昆明寰基生物芯片开发有限公司 | Specificity determination and application of one section of gonococcus DNA sequence |
| CN1465716A (en) * | 2002-06-26 | 2004-01-07 | 昆明寰基生物芯片开发有限公司 | Determination for specificity of one segmentum gonococcus DNA sequence (3) and use thereof |
| CN1465714A (en) * | 2002-06-26 | 2004-01-07 | 昆明寰基生物芯片开发有限公司 | One segmentum gonococcus DNA sequence (2) specificity detemination and use thereof |
| CN1465715A (en) * | 2002-06-26 | 2004-01-07 | 昆明寰基生物芯片开发有限公司 | Determination for specificity of one segmentum gonococcus DNA sequence (5) and use thereof |
| CN103060453A (en) * | 2013-01-10 | 2013-04-24 | 湖南圣湘生物科技有限公司 | Kit for detecting neisseria gonorrheae (NG) |
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Application publication date: 20150603 |
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| RJ01 | Rejection of invention patent application after publication |