CN101831488A - Method for quickly detecting gonorrhea Neisseria by utilizing loop-mediated isothermal amplification technology - Google Patents
Method for quickly detecting gonorrhea Neisseria by utilizing loop-mediated isothermal amplification technology Download PDFInfo
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Abstract
The invention belongs to biomedical diagnosis, and discloses a method for detecting gonorrhea Neisseria by utilizing a loop-mediated isothermal amplification technology and a sequence of a primer used by the method. By combining the loop-mediated isothermal amplification technology with the fluorochrome development, the method can realize quick isothermal nucleic acid amplification of the specific DNA sequence of gonorrhea Neisseria and result detection on the premise of not using a PCR amplifier, an electrophoresis apparatus or an ultraviolet gel imaging system. The method is simple to operate, and can be used for detecting the gonorrhea Neisseria in urethral secretions of a clinic outpatient or serves as a method for self-check of patients.
Description
Technical field
The invention belongs to the biomedical diagnostic field, be specifically related to a kind of method that is used to use the quick and convenient detection Diplococcus gonorrhoeae of ring mediated isothermal amplification (LAMP) technology.Can be used for clinical diagnosis and individual early screening etc.
Background technology
Diplococcus gonorrhoeae (Neisseria gonorrhoeae) is the common urogenital system pathogenic agent that spreads disease.Traditional clinical detection method is to use bacterial cultivation, but this method operation easier is big, and length consuming time, sample shipping conditions are very strict, and are depositing the low shortcoming of recall rate, cause failing to pinpoint a disease in diagnosis mistaken diagnosis easily.In addition, this method also needs the sample that obtains of intrusive mood, the patient is caused big painful.Conventional PCR detection method can be avoided this problem, and has the commercial reagents box that some methods of utilizing nucleic acid amplification detect Diplococcus gonorrhoeae, but these test kits all need to use specific professional instrument, need finish detection in the laboratory of specialty.The most important thing is that in addition conventional polymerase chain reaction technology (round pcr) is difficult to avoid fully false-positive problem.This is to need manual control switch PCR pipe and use pipettor to draw sample because PCR finishes electrophoresis, carries out agarose gel electrophoresis the result is judged.Because the high density of PCR product, the open pipe stopped pipe to the processing of its product to operator require highly, the careless slightly then pollution of product aerogel very easily causes the appearance of false positive results.In addition, use this technology to carry out the nucleic acid amplification amplification and need use whizzer, PCR instrument, electrophoresis apparatus, a series of professional molecular biology instruments such as gel imaging system.These instruments are all very expensive, need trained professional to operate, moreover, detection to the result determines also to need to use agarose gel electrophoresis, length not only consuming time, complex procedures is difficult to realize large sample operation, and needs operator that skilled molecular biology operative technique is arranged, and also needs operator's Long contact time carcinogens ethidium bromide etc. that health is had the toxic and harmful substance that has a strong impact on.
The another kind of technology of development afterwards, fluorescent quantitative PCR technique is measured the variation of PCR inner fluorescent tube value the result is carried out interpretation in the time of by use PCR reaction.Do not carry out the electrophoresis judged result owing to do not need open pipe to take out the PCR product, can avoid the false positive problem of PCR product aerogel crossed contamination to a certain extent.Some methods of using fluorescent quantitative PCR techniques to detect gonorrhoeas are also grown up, and disclose the test kit that a kind of fluorescent quantitative PCR technique that uses self cancellation probe detects gonorrhoea as patent CN 100415900c.Yet fluorescent quantitative PCR technique need use expensive quantitative real time PCR Instrument (the supreme million people people's coin of hundreds of thousands of easily), and this has limited being extensive use of of this technology, has only the laboratory of minority specialty just can carry out relevant detection.
Gonorrhoea is a kind of as venereal disease, generally by sex track transmission but and be not limited to spread through sex intercourse.But influence or some psychological reasons of traditional Chinese culture, some patients or suspection patient are unwilling or shyly go up regular hospital and check, and tendency looks for some subterranean informal itinerant doctors to carry out diagnoses and treatment.These underground itinerant doctor's technology are very different, and generally because the plant and instrument condition restriction, can only only depend on the experience judgement rather than use modern molecular biology technique to make a definite diagnosis, cause misdiagnosis rate rate of missed diagnosis height, not only may cause the financial loss of unnecessary huge stress to the patient, and may bring potential to threaten to public health owing to fail to pinpoint a disease in diagnosis the wider propagation that causes disease.If a kind of detection kit or the detection method that can buy in regular pharmacy fast arranged, can oneself finish the diagnoses and treatment suggestion that detects and science is provided, will bring very big help to the raising of public health level undoubtedly.
After 2000, occurred a kind of and the distinct technology of polymerase chain reaction technology for nucleic acid amplification technologies: loop-mediated isothermal amplification technique (loop-mediated isothermal amplification, LAMP).The mechanism that loop-mediated isothermal amplification technique has mainly utilized the Bst archaeal dna polymerase newly-generated nucleic acid strand can be replaced from new synthetic dna double chain is implemented under the invariable temperature nucleic acid is increased.About the detailed reaction mechanism of this technology with and with the different of polymerase chain reaction can be referring to patent CN 100422323C (grant number) and CN 101173317A (publication number).The existing at present academic article report that this technology is applied to the rapid detection of some diseases, as acquired immune deficiency syndrome (AIDS), the detection of singapore hemorrhagic fever even swine disease poison etc.But be applied to the detection of Diplococcus gonorrhoeae, do not appear in the newspapers yet both at home and abroad.
Summary of the invention
A kind of loop-mediated isothermal amplification technique (loop-mediated isothermal amplification, LAMP) method that gonorrhoea is detected utilized disclosed in the present invention.It is that the isothermal chain is replaced amplification that LMAP reacts topmost characteristics.This makes the miniature constant temperature device of a cheap and simple just can finish reaction.As the micro metal bath apparatus, small-sized water-bath device or small-sized baking oven.If make and supporting 65 degree constant temperature subminiature devices of lamp test kit even can be with the realization of more cheap ball bearing made using.Loop-mediated isothermal amplification technique need be provided with 4 primers or 6 primers to six positions of target gene, thereby utilize specific have the active hot resistant DNA polymerase Bst of strand displacement enzyme and under isothermal condition, form special construction and make target gene realize efficient amplification, and do not need to carry out traditional thermal cycling.Compare round pcr, the LAMP technology is more suitable in being applied to a large amount of samples, non-breadboard detection place.This technology not only needs professional instrument hardly, and is more simple in the operation, lower to operator's technical requirements, and the reaction times only needs about one hour.
Than the more or less freely design of primers of round pcr, the difficult point of LAMP technology is that it need design two long two short four special primers.Thereby the structure that target sequence is formed dumbbell shape that makes that it can be special realizes the amplification amplification that different alternately chains replaces that reacts completely with PCR.The final success or not that detects of the quality of design of primers and sequence specific sexual intercourse thereof.
The objective of the invention is to overcome the deficiency of at present existing gonorrhoea detection method, utilize loop-mediated isothermal amplification to realize a kind of gonorrhoea detection method that need not professional instrument fast.To achieve these goals, the present invention adopts following technical scheme.
The present invention proposes that for the first time loop-mediated isothermal amplification technique is applied to Diplococcus gonorrhoeae and detects.Specifically realize as follows: the cryptic plasmid dna fragmentation of selecting Diplococcus gonorrhoeae.The database (http://www.ncbi.nlm.nih.gov/) that its segmentation is committed on the American National biotechnology center website carries out Blast retrieval (webpage clicking upper left corner Blast button, and with fasta form submission sequence, select the nt option), seek the dna fragmentation that distinctive and other bacteriums of gonorrhoea do not have high homology by the blast program analysis.In the present invention, the contriver selects non-coding sequence between B albumen coded sequence on the stealthy plasmid and B albumen and the A albumen according to Blast result, carries out the LAMP design of primers voluntarily.The LAMP primers F 3 that provides five covers can be used for this position specific amplified in this programme, B3 and FIP BIP.This five covers primer all can carry out effective LAMP amplification to the purpose fragment on the Diplococcus gonorrhoeae.
The first cover primer
| ??F3 | ??TAACTCGGCTGCCAAGCT | ??SEQ?ID?NO：1 |
| ??B3 | ??AAAGCAACGCGGGTATGC | ??SEQ?ID?NO：2 |
| ??FIP | ??AACGCACTTGGGCGAAAGGTTCTGCTGCGCTAAACTCGT | ??SEQ?ID?NO：3 |
| ??F3 | ??TAACTCGGCTGCCAAGCT | ??SEQ?ID?NO：1 |
| ??BIP | ??TCATTCGCTGCTCGATTGCTGCGAAGCGAATGCTGGACGC | ??SEQ?ID?NO：4 |
The second cover primer
| ??F3 | ??AGCTGCTGCGCTAAACTC | ??SEQ?ID?NO：5 |
| ??B3 | ??AAATTGCCCAGTCGAGAACA | ??SEQ?ID?NO：6 |
| ??FIP | ??CAATCACGCAGCAATCGAGCAGCTGCTCTAGCTCTGCCAAC | ??SEQ?ID?NO：7 |
| ??BIP | ??AATTCCGCTAACGCGTCCAGCGCAAAGCAACGCGGGTAT | ??SEQ?ID?NO：8 |
The 3rd cover primer
| ??F3 | ??CGTGTTTTCCTGCTCTAGCT | ??SEQ?ID?NO：9 |
| ??B3 | ??AAATTGCCCAGTCGAGAACA | ??SEQ?ID?NO：10 |
| ??FIP | ??CAATCACGCAGCAATCGAGCAGGCCAACCTTTCGCCCAAG | ??SEQ?ID?NO：11 |
| ??BIP | ??AATTCCGCTAACGCGTCCAGCGCAAAGCAACGCGGGTAT | ??SEQ?ID?NO：12 |
The quadruplet primer
| ??F3 | ??TGCTGTTTCAAGTCGTCCAG | ??SEQ?ID?NO：13 |
| ??B3 | ??TGAAGCAAAGCGAGCAGAA | ??SEQ?ID?NO：14 |
| ??FIP | ??GGTATATCGGCGGCAGGGTTGCTCGTTCTTGACGCTCCAT | ??SEQ?ID?NO：15 |
| ??BIP | ??GCAAGCTCCACAGATAGGGCTGATATAAACGCCCGGCAGTT | ??SEQ?ID?NO：16 |
The 5th cover primer
| ??F3 | ??TGAACAGCCCTGCTATGACT | ??SEQ?ID?NO：17 |
| ??B3 | ??CAGACATCACGCACCGAAG | ??SEQ?ID?NO：18 |
| ??FIP | ??GCCCGGCAGTTACGCATGAGTACCTAGCAAGCTCCACAGA | ??SEQ?ID?NO：19 |
| ??BIP | ??CGCTTTGCTTCAATGCCTCGTTGCCAGCATAGAGCAACAAAC | ??SEQ?ID?NO：20 |
More than five cover primers be used for non-coding sequence between LAMP method amplification Diplococcus gonorrhoeae B albumen coded sequence and B albumen and the A albumen.Entrusting Shanghai living worker company to carry out primer primer sequence synthesizes.
Its positive control is selected following fragment on the Diplococcus gonorrhoeae cryptic plasmid:
CATTCCCCATCCCCTGCTTTGGGTTCGTTTGTATCGTTGGCTTATCGTTTGGCTGGTTGATTCAAGATTTCGCTCTGCCGTTGCCGTATTTCGCTCTGCCGCTCTAACTCGGCTGCCAAGCTCGCTAGCTGCTGCGCTAAACTCGTGTTTTCCTGCTCTAGCTCTGCCAACCTTTCGCCCAAGTGCGTTAAGGCTTTCATCATTCGCTGCTCGATTGCTGCGTGATTGCTCTCTAATTCCGCTAACGCGTCCAGCATTCGCTTCTCGGTCGCTACGCATACCCGCGTTGCTTTGCTGTTCTCGACTGGGCAATTTTCCAGTGTCAAACCTTTGGTCTTGGTTTCCAACAGGTCTAGGGTGCGCTCTGCTTCGGCTCTCTGCTGTTTCAAGTCGTCCAGCTCGTTCTTGACGCTCCATATCGCTATGAACAGCCCTGCTATGACTATCAACCCTGCCGCCGATATACCTAGCAAGCTCCACAGATAGGGCTTGAATACTGCCTTGCTCATGCGTAACTGCCGGGCGTTTATATCGGCGGTTATTTTCTGCTCGCTTTGCTTCAATGCCTCGTTGATATTTTTCCGTAACGTCTCTAAGTCTGCTTTCGTTTGTTGCTCTATGCTGGCGGCTTCGGTGCGTGATGTCTGCTCGAAGGTCTTCGCCAAATCGGAAATCTTGCTCATACAGTGCGCCTTTCAGTCGGATGTTGCGCCCTTTTGGGTCCGGGTTCTTGATGCTGATGCTGCTGATGGTCGCTCGTGATATTTCAAAACCTACCTTTTCCAGCGTTTCCAGCACGTCTGCGCGGCTTTTTAGCTTGCCTGATAGGGCTAGGGCTTCTAAGCCGTCTGTGATGCTCTGTGATGCTCTGTGCGGCTTCCTGCGTGTTTCTCGGCAGGTCTTTGGCTTGGGTCATGCTCTGCCGTTTGGCGGGGTCGTCTGGGTCGCTGTATCCGTGCGTCAGGTTCTGCATGGTGCGCCATGCGTCCACTCTTCCTCTGTCGGCGGCG????????????????????????????SEQ?ID?NO：33
Use round pcr to prepare positive template, reaction conditions is as follows:
The design primer is as follows, and entrusts Shanghai to give birth to worker company and synthesize.
Forward primer 5 ' CATTCCCCATCCCCTGCTTTGGGTT 3 ' SEQ ID NO:29
Reverse primer 5 ' GCCGCCGACAGAGGAAGAGTGGACG 3 ' SEQ ID NO:30
The PCR system is as follows:
| The total DNA of Diplococcus gonorrhoeae | ??1μl |
| ??dNTP | ??0.5μl |
| Forward primer | ??2μl |
| Reverse primer | ??2μl |
| PCR reaction buffer (Takara company) | ??5μl |
| Deionized water | ??40μl |
| LA Taq enzyme (Takara company) | ??0.5μl |
Above-mentioned all the components is added to a little PCR pipe, put into the PCR instrument, it is as follows that the PCR instrument is provided with reaction conditions:
Step 1 94 degree 2 minutes
Step 2 94 degree 1 minute, then
54 degree 1 minute, then
72 degree 1 minute
Step 3 repeating step 2 32 times.
Confirm entrusting the order-checking of Beijing China major company after the gained PCR product electrophoresis.Subsequently again to confirm that the total DNA of Diplococcus gonorrhoeae that the PCR product is replaced in the above-mentioned PCR system as standard form carries out the multitube pcr amplification.Use Minielute PCR purification kit (QIAGEN company product) to carry out purifying the per 50 μ l of resulting product, and be diluted to 0.5ml, promptly make positive over against shining template.0.5ml available 1000 times of template is used 0.5 μ l at every turn.
Because the infection population of international reports Diplococcus gonorrhoeae diverse geographic location all over the world has different strain systems, and the discovery report of the Diplococcus gonorrhoeae of ccpB gene fragment disappearance was arranged on Australia and other places.For differentiating this sudden change Diplococcus gonorrhoeae, the present invention has also designed 2 simultaneously and has overlapped the LAMP primer that is used for 16S rRNA coding region.Because how bacterium has higher homology (98%) for 16S rRNA district blast analytical results and meninx, may cause to intersect and increase, this sudden change Diplococcus gonorrhoeae is seldom found report in China simultaneously.So these following two only conduct standby contrasts in particular cases of cover primers.
The 6th cover primer
| ??F3 | ??CGCGCCCACCTTAAACAG | ??SEQ?ID?NO：21 |
| ??B3 | ??CCGTTGCGTCAAAAGTCAAC | ??SEQ?ID?NO：22 |
| ??FIP | ??ATTTGTCGAAGACGCGCTGCGCTTCAATTTTTGCGCGGTCG | ??SEQ?ID?NO：23 |
| ??BIP | ??GCCCCTTCTTAGGGACGCAAGTCCAATCCTACGGCGTTAC | ??SEQ?ID?NO：24 |
The 7th cover primer
| ??F3 | ??GCCCACCTTAAACAGCTTCA | ??SEQ?ID?NO：25 |
| ??B3 | ??CCGTTGCGTCAAAAGTCAAC | ??SEQ?ID?NO：26 |
| ??FIP | ??GTGGCAATTTGTCGAAGACGCGGCGGTCGGTTCAAAACTGT | ??SEQ?ID?NO：27 |
| ??BIP | ??GCCCCTTCTTAGGGACGCAAGTCCAATCCTACGGCGTTAC | ??SEQ?ID?NO：28 |
Its positive control is selected following 16S rRNA encoding sequence:
ATCCTGGCTCAGATTGAACGCTGGCGGCATGCTTTACACATGCAAGTCGGACGGCAGCACAGGGAAGCTTGCTTCTCGGGTGGCGAGTGGCGAACGGGTGAGTAACATATCGGAACGTACCGGGTAGCGGGGGATAACTGATCGAAAGATCAGCTAATACCGCATACGTCTTGAGAGGGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCCGAGCGGCCGATATCTGATTAGCTGGTTGGCGGGGTAAAGGCCCACCAAGGCGACGATCAGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGTCTGAAGAAGGCCTTCGGGTTGTAAAGGACTTTTGTCAGGGAAGAAAAGGCTGTTGCCAATATCGGCGGCCGATGACGGTACCTGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGGGCGCAGACGGTTACTTAAGCAGGATGTGAAATCCCCGGGCTCAACCCGGGAACTGCGTTCTGAACTGGGTGACTCGAGTGTGTCAGAGGGAGGTGGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCTCCTGGGATAACACTGACGTTCATGTCCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAATTAGCTGTTGGGCAACTTGATTGCTTGGTAGCGTAGCTAACGCGTGAAATTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGTTTTGACATGTGCGGAATCCTCCGGAGACGGAGGAGTGCCTTCGGGAGCCGTAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCCATCATTCGGTTGGGCACTCTAATGAGACTGCCGGTGACAAGCCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGACCAGGGCTTCACACGTCATACAATGGTCGGTACAGAGGGTAGCCAAGCCGCGAGGCGGAGCCAATCTCACAAAACCGATCGTAGTCCGGATTGCACTCTGCAACTCGAGTGCATGAAGTCGGAATCGCTAGTAATCGCAGGTCAGCATACTGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGGGATACCAGAAGTAGGTAGGGTAACCGCAAGGAGTCCGCTTACCACGGTATGCTTCATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGGAACCTGCGGCTGGATCACCT?????????????????????????????????????????????SEQ?ID?NO：34
Use round pcr to prepare positive template, reaction conditions is as follows:
The design primer is as follows, and entrusts Shanghai to give birth to worker company and synthesize.
Forward primer 5 ' ATCCTGGCTCAGATTGAACGCTGGC 3 ' SEQ ID NO:31
Reverse primer 5 ' CCGCAGGTTCCCCTACGGCTACCTT 3 ' SEQ ID NO:32
The PCR system is as follows:
| The total DNA of Diplococcus gonorrhoeae | ??1μl |
| ??dNTP | ??0.5μl |
| Forward primer | ??2μl |
| Reverse primer | ??2μl |
| PCR reaction buffer (Takara company) | ??5μl |
| Deionized water | ??40μl |
| LA Taq enzyme (Takara company) | ??0.5μl |
Above-mentioned all the components added good put into the PCR instrument to a little PCR pipe, it is as follows that the PCR instrument is provided with reaction conditions:
Step 1 94 degree 2 minutes
Step 2 94 degree 1 minute, then
54 degree 1 minute, then
72 degree 2 minutes
Step 3 repeating step 2 32 times.
Confirm entrusting the order-checking of Beijing China major company after the gained PCR product electrophoresis.The total DNA of Diplococcus gonorrhoeae that replaces in the above-mentioned PCR system as standard form with the PCR product of affirmation again carries out pcr amplification subsequently.Use Minielute PCR purification kit (QIAGEN company product) to carry out purifying the per 50 μ l of resulting product, and be diluted to 0.5ml, promptly make the positive control template.0.5ml available 1000 times of template is used 0.5 μ l at every turn.
Below further set forth the present invention with embodiment.Wherein used sample is urethral secretions or urine.Because the height sensitivity and the specificity of LAMP amplification technique have been avoided traditional intrusive mood to get the urethra sample and have been cultivated the misery of bringing to the patient.Make present method become and be the special non-invasive diagnosis method of a kind of sensitivity.
Description of drawings
Fig. 1: show embodiment with the fluorexon judged result.
Fig. 2: show embodiment with SYBR green judged result.
Embodiment
Be described below with regard to hospital's clinical implementation mode and patient oneself examination embodiment respectively below.
Hospital's embodiment:
Use QIAamp DNA Mini Kit (QIAGEN company) to patient's urethral secretions or urine centrifugation sample extraction high purity DNA, concrete operations are finished according to its specification sheets.(this step also can use the similar DNA extraction test kit of other biological company to finish).Use the mentioned reagent box to reclaim and obtain gonorrhoea patient urethral secretions DNA sample 50 μ l, be labeled as sample 2; And healthy human urine road secretory product DNA sample 50 μ l are labeled as sample 1.From sample 1 and sample 2, respectively take out the new little PCR pipe of 2 μ l to, add 50 μ l reaction solutions (composition is as follows) again.Shut PCR pipe lid.Pipe is put into 65 degree thermostat metals bathe insulation 1 hour.
Subsequently pipe is taken out visual inspection liquid in pipe color.If liquid becomes green in the pipe, special nucleotide sequence then is described, Diplococcus gonorrhoeae ccpB sheet segment DNA is effectively increased, and the amplification sample contains the ccpB fragment after the amplification of a large amount of Diplococcus gonorrhoeae.If liquid color is constant, then do not contain the thymus nucleic acid of Diplococcus gonorrhoeae in the interpret sample.In this experiment, as shown in accompanying drawing, sample 1 liquid in pipe still is clear, colorless (with fluorexon as developer) or orange red (with SYBR green as developer), the thymus nucleic acid that does not contain the gonorrhoea bacterium in the initial detecting sample is described, promptly this sample supplier is not by infection due to Neisseria gonorrhoeae.And the liquid in the sample 2 becomes green, and the thymus nucleic acid that wherein contains the gonorrhoea bacterium is described, promptly this sample supplier is by infection due to Neisseria gonorrhoeae.
The reaction solution composition, wherein four primers can use any cover in above-mentioned one to the five cover primer.
| ??Tris?buffer(pH?8.8) | ??200mM |
| ??KCl | ??30mM |
| ??MgSO 4 | ??30mM |
| ??(NH 4) 2SO 4 | ??20mM |
| ??Tween?20 | ??0.2％ |
| ??Betaine | ??1.6M |
| ??Tris?buffer(pH?8.8) | ??200mM |
| ??dNTPs | ??4mM |
| ??MnCl 2 | ??1mM |
| Fluorescent indicator fluorexon (perhaps SYBR green) | ??50mM |
| Primers F IP | ??50pmol |
| Primer BIP | ??50pmol |
| Primers F 3 | ??15pmol |
| Primer B3 | ??15pmol |
| The Bst archaeal dna polymerase | ??20U |
Above reaction solution composition all is stored in negative 20 degree refrigerators.
Patient's self check embodiment:
Get the gonorrhoea patient and urinate 100 μ l (section urine at the beginning of early morning), be labeled as sample 2; And healthy human urine 100 μ l (section urine at the beginning of early morning) are labeled as sample 1.From sample 1 and sample 2, respectively take out the little PCR pipe of the new 0.5ml of 2 μ l to, add 5 μ l reaction solutions (composition is as follows) again.Shut PCR pipe lid.Pipe is put into 65 degree constant temperature micro metals bathe insulation 1 hour.
Subsequently pipe is taken out, if liquid becomes green in the visual inspection pipe, special nucleotide sequence is described then, Diplococcus gonorrhoeae ccpB sheet segment DNA is effectively increased, and the amplification sample contains the ccpB fragment after the amplification of a large amount of Diplococcus gonorrhoeae.If liquid color is constant, then do not contain the thymus nucleic acid of Diplococcus gonorrhoeae in the interpret sample.In this experiment, as shown in accompanying drawing, sample 1 liquid in pipe still is that sample 1 liquid in pipe still is clear, colorless (with fluorexon as developer) or orange red (with SYBR green as developer), the thymus nucleic acid that does not contain the gonorrhoea bacterium in the initial detecting sample is described, promptly this sample supplier is not by infection due to Neisseria gonorrhoeae.And the liquid in the sample 2 becomes green, and the thymus nucleic acid that wherein contains the gonorrhoea bacterium is described, promptly this sample supplier is by infection due to Neisseria gonorrhoeae.
The reaction solution composition, wherein four primers can use any cover in above-mentioned one to the five condom primer.
| Tris damping fluid (pH 8.8) | ??200mM |
| ??KCl | ??30mM |
| ??MgSO 4 | ??30mM |
| ??(NH 4) 2SO 4 | ??20mM |
| ??Tween?20 | ??0.2％ |
| Trimethyl-glycine | ??1.6M |
| Tris damping fluid (pH 8.8) | ??200mM |
| ??dNTPs | ??4mM |
| ??MnCl 2 | ??1mM |
| Fluorescent indicator fluorexon (perhaps SYBR green) | ??50mM |
| Primers F IP | ??50pmol |
| Primer BIP | ??50pmol |
| Primers F 3 | ??15pmol |
| Primer B3 | ??15pmol |
| The Bst archaeal dna polymerase | ??20U |
In the test kit pattern of patient's self check,
| Tris damping fluid (pH 8.8) | ??200mM |
| ??KCl | ??30mM |
| ??MgSO 4 | ??30mM |
| ??(NH 4) 2SO 4 | ??20mM |
| ??Tween?20 | ??0.2％ |
| Trimethyl-glycine | ??1.6M |
| ??dNTPs | ??4mM |
| ??MnCl 2 | ??1mM |
| Fluorescent indicator fluorexon (perhaps SYBR green) | ??50mM |
Form with liquid exists, and is labeled as solution A.
And following composition
| Primers F IP | ??50pmol |
| Primer BIP | ??50pmol |
| Primers F IP | ??50pmol |
| Primers F 3 | ??15pmol |
| Primer B3 | ??15pmol |
| The Bst archaeal dna polymerase | ??20U |
Form with lyophilized powder is being deposited, and is labeled as powder B.
The form that solution A and powder B all can seal is kept at room temperature.But the long-term placement of powder B still need be kept at negative 20 degree refrigerators to avoid enzyme deactivation.Use preceding with solution A and powder B mixing.Can obtain reaction solution.Simultaneously in test kit, provide small-sized pipettor with little pipette form.To be the patient draw minor quantities of urine 2 μ l with this pipettor adds and can begin in the above-mentioned mixed solution to detect.
Sequence table
＜110〉Sun Xingjiang
＜120〉use loop-mediated isothermal amplification technique rapid detection Diplococcus gonorrhoeae
<160>34
<170>PatentIn?version?3.3
<210>1
<211>18
<212>DNA
＜213〉primer
<400>1
taactcggct?gccaagct?????????????????????????????????????????????18
<210>2
<211>18
<212>DNA
＜213〉primer
<400>2
aaagcaacgc?gggtatgc?????????????????????????????????????????????18
<210>3
<211>39
<212>DNA
＜213〉primer
<400>3
aacgcacttg?ggcgaaaggt?tctgctgcgc?taaactcgt??????????????????????39
<210>4
<211>40
<212>DNA
＜213〉primer
<400>4
tcattcgctg?ctcgattgct?gcgaagcgaa?tgctggacgc?????????????????????40
<210>5
<211>18
<212>DNA
＜213〉primer
<400>5
agctgctgcg?ctaaactc???????????????????????????????????????18
<210>6
<211>20
<212>DNA
＜213〉primer
<400>6
aaattgccca?gtcgagaaca?????????????????????????????????????20
<210>7
<211>41
<212>DNA
＜213〉primer
<400>7
caatcacgca?gcaatcgagc?agctgctcta?gctctgccaa?c?????????????41
<210>8
<211>39
<212>DNA
＜213〉primer
<400>8
aattccgcta?acgcgtccag?cgcaaagcaa?cgcgggtat????????????????39
<210>9
<211>20
<212>DNA
＜213〉primer
<400>9
cgtgttttcc?tgctctagct?????????????????????????????????????20
<210>10
<211>20
<212>DNA
＜213〉primer
<400>10
aaattgccca?gtcgagaaca??????????????????????????????????20
<210>11
<211>40
<212>DNA
＜213〉primer
<400>11
caatcacgca?gcaatcgagc?aggccaacct?ttcgcccaag????????????40
<210>12
<211>39
<212>DNA
＜213〉primer
<400>12
aattccgcta?acgcgtccag?cgcaaagcaa?cgcgggtat?????????????39
<210>13
<211>20
<212>DNA
＜213〉primer
<400>13
tgctgtttca?agtcgtccag??????????????????????????????????20
<210>14
<211>19
<212>DNA
＜213〉primer
<400>14
tgaagcaaag?cgagcagaa????????????????????????????????19
<210>15
<211>40
<212>DNA
＜213〉primer
<400>15
ggtatatcgg?cggcagggtt?gctcgttctt?gacgctccat?????????40
<210>16
<211>41
<212>DNA
＜213〉primer
<400>16
gcaagctcca?cagatagggc?tgatataaac?gcccggcagt?t???????41
<210>17
<211>20
<212>DNA
＜213〉primer
<400>17
tgaacagccc?tgctatgact???????????????????????????????20
<210>18
<211>19
<212>DNA
＜213〉primer
<400>18
cagacatcac?gcaccgaag????????????????????????????????19
<210>19
<211>40
<212>DNA
＜213〉primer
<400>19
gcccggcagt?tacgcatgag?tacctagcaa?gctccacaga?????????????????????40
<210>20
<211>42
<212>DNA
＜213〉primer
<400>20
cgctttgctt?caatgcctcg?ttgccagcat?agagcaacaa?ac??????????????????42
<210>21
<211>18
<212>DNA
＜213〉primer
<400>21
cgcgcccacc?ttaaacag?????????????????????????????????????????????18
<210>22
<211>20
<212>DNA
＜213〉primer
<400>22
ccgttgcgtc?aaaagtcaac???????????????????????????????????????????20
<210>23
<211>41
<212>DNA
＜213〉primer
<400>23
atttgtcgaa?gacgcgctgc?gcttcaattt?ttgcgcggtc?g???????????????????41
<210>24
<211>40
<212>DNA
＜213〉primer
<400>24
gccccttctt?agggacgcaa?gtccaatcct?acggcgttac?????????????40
<210>25
<211>20
<212>DNA
＜213〉primer
<400>25
gcccacctta?aacagcttca???????????????????????????????????20
<210>26
<211>20
<212>DNA
＜213〉primer
<400>26
ccgttgcgtc?aaaagtcaac???????????????????????????????????20
<210>27
<211>41
<212>DNA
＜213〉primer
<400>27
gtggcaattt?gtcgaagacg?cggcggtcgg?ttcaaaactg?t???????????41
<210>28
<211>40
<212>DNA
＜213〉primer
<400>28
gccccttctt?agggacgcaa?gtccaatcct?acggcgttac?????????????40
<210>29
<211>25
<212>DNA
＜213〉primer
<400>29
cattccccat?cccctgcttt?gggtt???????????????????????????????????????25
<210>30
<211>25
<212>DNA
＜213〉primer
<400>30
gccgccgaca?gaggaagagt?ggacg???????????????????????????????????????25
<210>31
<211>25
<212>DNA
＜213〉primer
<400>31
atcctggctc?agattgaacg?ctggc???????????????????????????????????????25
<210>32
<211>25
<212>DNA
＜213〉primer
<400>32
ccgcaggttc?ccctacggct?acctt???????????????????????????????????????25
<210>33
<211>1010
<212>DNA
<213>Neisseria?gonorrhoeae
<400>33
cattccccat?cccctgcttt?gggttcgttt?gtatcgttgg?cttatcgttt?ggctggttga????60
ttcaagattt?cgctctgccg?ttgccgtatt?tcgctctgcc?gctctaactc?ggctgccaag????120
ctcgctagct?gctgcgctaa?actcgtgttt?tcctgctcta?gctctgccaa?cctttcgccc????180
aagtgcgtta?aggctttcat?cattcgctgc?tcgattgctg?cgtgattgct?ctctaattcc????240
gctaacgcgt?ccagcattcg?cttctcggtc?gctacgcata?cccgcgttgc?tttgctgttc????300
tcgactgggc?aattttccag?tgtcaaacct?ttggtcttgg?tttccaacag?gtctagggtg????360
cgctctgctt?cggctctctg?ctgtttcaag?tcgtccagct?cgttcttgac?gctccatatc????420
gctatgaaca?gccctgctat?gactatcaac?cctgccgccg?atatacctag?caagctccac????480
agatagggct?tgaatactgc?cttgctcatg?cgtaactgcc?gggcgtttat?atcggcggtt????540
attttctgct?cgctttgctt?caatgcctcg?ttgatatttt?tccgtaacgt?ctctaagtct????600
gctttcgttt?gttgctctat?gctggcggct?tcggtgcgtg?atgtctgctc?gaaggtcttc????660
gccaaatcgg?aaatcttgct?catacagtgc?gcctttcagt?cggatgttgc?gcccttttgg????720
gtccgggttc?ttgatgctga?tgctgctgat?ggtcgctcgt?gatatttcaa?aacctacctt????780
ttccagcgtt?tccagcacgt?ctgcgcggct?ttttagcttg?cctgataggg?ctagggcttc????840
taagccgtct?gtgatgctct?gtgatgctct?gtgcggcttc?ctgcgtgttt?ctcggcaggt????900
ctttggcttg?ggtcatgctc?tgccgtttgg?cggggtcgtc?tgggtcgctg?tatccgtgcg????960
tcaggttctg?catggtgcgc?catgcgtcca?ctcttcctct?gtcggcggcg???????????????1010
<210>34
<211>1521
<212>DNA
<213>Neisseria?gonorrhoeae
<400>34
atcctggctc?agattgaacg?ctggcggcat??gctttacaca?tgcaagtcgg?acggcagcac???60
agggaagctt?gcttctcggg?tggcgagtgg?cgaacgggtg?agtaacatat?cggaacgtac????120
cgggtagcgg?gggataactg?atcgaaagat?cagctaatac?cgcatacgtc?ttgagaggga????180
aagcagggga?ccttcgggcc?ttgcgctatc?cgagcggccg?atatctgatt?agctggttgg????240
cggggtaaag?gcccaccaag?gcgacgatca?gtagcgggtc?tgagaggatg?atccgccaca????300
ctgggactga?gacacggccc?agactcctac?gggaggcagc?agtggggaat?tttggacaat????360
gggcgcaagc?ctgatccagc?catgccgcgt?gtctgaagaa?ggccttcggg?ttgtaaagga????420
cttttgtcag?ggaagaaaag?gctgttgcca?atatcggcgg?ccgatgacgg?tacctgaaga????480
ataagcaccg?gctaactacg?tgccagcagc?cgcggtaata?cgtagggtgc?gagcgttaat????540
cggaattact?gggcgtaaag?cgggcgcaga?cggttactta?agcaggatgt?gaaatccccg????600
ggctcaaccc?gggaactgcg?ttctgaactg?ggtgactcga?gtgtgtcaga?gggaggtgga????660
attccacgtg?tagcagtgaa?atgcgtagag?atgtggagga?ataccgatgg?cgaaggcagc????720
ctcctgggat?aacactgacg?ttcatgtccg?aaagcgtggg?tagcaaacag?gattagatac????780
cctggtagtc?cacgccctaa?acgatgtcaa?ttagctgttg?ggcaacttga?ttgcttggta????840
gcgtagctaa?cgcgtgaaat?tgaccgcctg?gggagtacgg?tcgcaagatt?aaaactcaaa????900
ggaattgacg?gggacccgca?caagcggtgg?atgatgtgga?ttaattcgat?gcaacgcgaa????960
gaaccttacc?tggttttgac?atgtgcggaa?tcctccggag?acggaggagt?gccttcggga????1020
gccgtaacac?aggtgctgca?tggctgtcgt?cagctcgtgt?cgtgagatgt?tgggttaagt????1080
cccgcaacga?gcgcaaccct?tgtcattagt?tgccatcatt?cggttgggca?ctctaatgag????1140
actgccggtg?acaagccgga?ggaaggtggg?gatgacgtca?agtcctcatg?gcccttatga????1200
ccagggcttc?acacgtcata?caatggtcgg?tacagagggt?agccaagccg?cgaggcggag????1260
ccaatctcac?aaaaccgatc?gtagtccgga?ttgcactctg?caactcgagt?gcatgaagtc????1320
ggaatcgcta?gtaatcgcag?gtcagcatac?tgcggtgaat?acgttcccgg?gtcttgtaca????1380
caccgcccgt?cacaccatgg?gagtggggga?taccagaagt?aggtagggta?accgcaagga????1440
gtccgcttac?cacggtatgc?ttcatgactg?gggtgaagtc?gtaacaaggt?agccgtaggg????1500
gaacctgcgg?ctggatcacc?t??????????????????????????????????????????????1521
Claims (3)
1. a use loop-mediated isothermal amplification technique (LAMP technology) detects the method for Diplococcus gonorrhoeae.It is characterized in that,, utilize the LAMP technology that the isothermal nucleic acid amplification is carried out in the specific dna region territory of Diplococcus gonorrhoeae by using specific primer.From molecular level Diplococcus gonorrhoeae is detected or early screening.Wherein said special primer is selected from:
SEQ ID NO:1 to SEQ ID NO:28
2. loop-mediated isothermal amplification technique according to claim 1 (LAMP technology) detects the method for Diplococcus gonorrhoeae, it is characterized in that, arbitrary temperature is reacted between use 60 to 65 degree.
3. use loop-mediated isothermal amplification technique according to claim 1 (LAMP technology) detects the method for Diplococcus gonorrhoeae, it is characterized in that, uses fluorexon or ds DNA binding fluorescent dyes SYBR green that the result is carried out naked eyes and as seen develops the color.
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| CN200910037773A CN101831488A (en) | 2009-03-11 | 2009-03-11 | Method for quickly detecting gonorrhea Neisseria by utilizing loop-mediated isothermal amplification technology |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910037773A CN101831488A (en) | 2009-03-11 | 2009-03-11 | Method for quickly detecting gonorrhea Neisseria by utilizing loop-mediated isothermal amplification technology |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102230019A (en) * | 2011-07-08 | 2011-11-02 | 山东省农业科学院畜牧兽医研究所 | Primer of loop-mediated isothermal amplification (LAMP) for detecting multidrug-resistant cfr gene as well as kit and method for detecting multidrug-resistant cfr gene |
| WO2016023397A1 (en) * | 2014-08-15 | 2016-02-18 | 广州医科大学附属第三医院 | Primer group for gonococci detection, kit comprising primer group and uses thereof |
| WO2018089945A1 (en) * | 2016-11-10 | 2018-05-17 | Slipchip Corporation | Polynucleotides for the amplification and detection of neisseria gonorrhoeae |
| US10954572B2 (en) | 2019-07-25 | 2021-03-23 | Talis Biomedical Corporation | Polynucleotides for the amplification and detection of Neisseria gonorrhoeae |
| CN114107448A (en) * | 2022-01-27 | 2022-03-01 | 天津喜诺生物医药有限公司 | LAMP detection method based on Calcein fluorescence curve |
| US11326214B2 (en) | 2018-05-09 | 2022-05-10 | Talis Biomedical Corporation | Polynucleotides for the amplification and detection of chlamydia trachomatis |
| US11891662B2 (en) | 2019-12-02 | 2024-02-06 | Talis Biomedical Corporation | Polynucleotides for amplification and detection of human beta actin |
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2009
- 2009-03-11 CN CN200910037773A patent/CN101831488A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102230019A (en) * | 2011-07-08 | 2011-11-02 | 山东省农业科学院畜牧兽医研究所 | Primer of loop-mediated isothermal amplification (LAMP) for detecting multidrug-resistant cfr gene as well as kit and method for detecting multidrug-resistant cfr gene |
| CN102230019B (en) * | 2011-07-08 | 2012-12-19 | 山东省农业科学院畜牧兽医研究所 | Primer of loop-mediated isothermal amplification (LAMP) for detecting multidrug-resistant cfr gene as well as kit and method for detecting multidrug-resistant cfr gene |
| WO2016023397A1 (en) * | 2014-08-15 | 2016-02-18 | 广州医科大学附属第三医院 | Primer group for gonococci detection, kit comprising primer group and uses thereof |
| WO2018089945A1 (en) * | 2016-11-10 | 2018-05-17 | Slipchip Corporation | Polynucleotides for the amplification and detection of neisseria gonorrhoeae |
| US11326214B2 (en) | 2018-05-09 | 2022-05-10 | Talis Biomedical Corporation | Polynucleotides for the amplification and detection of chlamydia trachomatis |
| US10954572B2 (en) | 2019-07-25 | 2021-03-23 | Talis Biomedical Corporation | Polynucleotides for the amplification and detection of Neisseria gonorrhoeae |
| US11891662B2 (en) | 2019-12-02 | 2024-02-06 | Talis Biomedical Corporation | Polynucleotides for amplification and detection of human beta actin |
| CN114107448A (en) * | 2022-01-27 | 2022-03-01 | 天津喜诺生物医药有限公司 | LAMP detection method based on Calcein fluorescence curve |
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