CN104911275B - A kind of bacterial vaginitis detection kit - Google Patents
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- 238000001514 detection method Methods 0.000 title claims abstract description 26
- 208000004926 Bacterial Vaginosis Diseases 0.000 title claims abstract description 22
- 239000011535 reaction buffer Substances 0.000 claims abstract description 12
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 10
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 10
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 10
- 239000000980 acid dye Substances 0.000 claims abstract description 7
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 239000013642 negative control Substances 0.000 claims description 5
- 239000013641 positive control Substances 0.000 claims description 5
- 238000007400 DNA extraction Methods 0.000 claims description 3
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 3
- 238000012360 testing method Methods 0.000 abstract description 8
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 abstract description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 abstract description 2
- 241000207201 Gardnerella vaginalis Species 0.000 description 17
- 238000006243 chemical reaction Methods 0.000 description 16
- 238000000034 method Methods 0.000 description 11
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical group [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- 230000003321 amplification Effects 0.000 description 5
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- 238000005516 engineering process Methods 0.000 description 4
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- 241000725303 Human immunodeficiency virus Species 0.000 description 3
- 206010046914 Vaginal infection Diseases 0.000 description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- 102100021971 Bcl-2-interacting killer Human genes 0.000 description 1
- 208000004145 Endometritis Diseases 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 101000970576 Homo sapiens Bcl-2-interacting killer Proteins 0.000 description 1
- 101000846284 Homo sapiens Pre-mRNA 3'-end-processing factor FIP1 Proteins 0.000 description 1
- 238000007397 LAMP assay Methods 0.000 description 1
- 239000012807 PCR reagent Substances 0.000 description 1
- 241000191992 Peptostreptococcus Species 0.000 description 1
- 102100031755 Pre-mRNA 3'-end-processing factor FIP1 Human genes 0.000 description 1
- 208000006399 Premature Obstetric Labor Diseases 0.000 description 1
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- 230000002159 abnormal effect Effects 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- BCTILSWHJRTUIE-UHFFFAOYSA-N azanium;4-[4-[bis[4-(dimethylamino)phenyl]-hydroxymethyl]-3-methyl-5-oxo-4h-pyrazol-1-yl]benzenesulfonate Chemical compound [NH4+].C1=CC(N(C)C)=CC=C1C(O)(C=1C=CC(=CC=1)N(C)C)C1C(=O)N(C=2C=CC(=CC=2)S([O-])(=O)=O)N=C1C BCTILSWHJRTUIE-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
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Classifications
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- 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
- C12Q1/6876—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
-
- 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
- C12Q2600/158—Expression markers
Abstract
The present invention relates to a kind of bacterial vaginitis LAMP detection kit, does it comprise primer, reaction buffer, Bst? archaeal dna polymerase and nucleic acid dye.Use test kit of the present invention to carry out LAMP and detect the Oupatient Check-UPS that can meet bacterial vaginitis better and emergent or Site Detection to the fast and convenient property requirement of detection method.
Description
Technical field
The present invention relates to microorganism detection field, specifically, the present invention relates to a kind of bacterial vaginitis detection kit.
Background technology
Bacterial vaginitis (bacterialvaginosis, be called for short BV) be a kind of common, complicated, change into feature with normal vaginal microbial flora clinical syndrome, it can cause serious complication, comprise trachelitis, endometritis, also make patient increase to carry HIV and the risk of premature labor.In the past due to limited to its understanding, once reported a lot of title, as nonspecific vaginitis, Gardnerella vaginitis, vaginitis hemoptulus vaginalis etc.1984, its definite designation was bacterial vaginosis by Sweden's special topic International Academic Conference.BV is the modal vaginal infection diseases of the women of child-bearing age, and the morbidity in Out-patient Clinic of Department of Gynecology is 18.63% ~ 20%.Patient bears huge private sorrow, and family social economical load is heavy.
Gardnerella vaginalis (GardnerellaVaginalis) has comparative advantage in the microorganism species of BV, and content, apparently higher than other abnormal microflora, is the infector of BV.The expression of latest report gardnerella vaginalis energy obvious stimulation HIV virus in monocytes/macrophages system and T lymphocyte, increases relevant with the spreading rate of HIV.Therefore, check Samples subjects's medial vagina Gardnerella whether to exist, the morning for BV examines, early control and prevent most important.
At present method of direct smear, gram staining method, isolated culture, immunofluorescence technique etc. are mainly contained to the laboratory diagnosis of gardnerella vaginalis.And be in the detection that also starts to be applied to gardnerella vaginalis gradually of the molecular biology for detection of representative and BV diagnosis with nucleic acid amplification technologies.Wherein, ring mediated constant temperature nucleic acid amplification technology (loop-mediatedisothermalamplificationofDNA, LAMP) be that a kind of novel nucleic acid amplification method grown up in recent years is (see document NotomiT, OkayamaH, MasubuchiH, YonekawaT, WatanabeK, AminoN, HaseT.Loop-mediatedisothermalamplificationofDNA.NucleicA cidsRes.2000Jun15; 28 (12): E63.), be characterized in 6 zone design 4 species-specific primers for target gene, constant-temperature amplification under the effect of strand displacement archaeal dna polymerase, realizes the nucleic acid amplification of a large amount of copy number fast.Compared with temperature varied cyclical amplification technique, the demand of isothermal amplification technique to plant and instrument comparatively simplifies, simultaneously because reaction does not need denaturation renaturation process, and thus can effective Reaction time shorten.These characteristics makes LAMP technology can meet bacterial vaginitis Oupatient Check-UPS and emergent or Site Detection better to the fast and convenient property requirement of detection method.
Summary of the invention
The object of the present invention is to provide a kind of for bacterial vaginitis LAMP detection kit.
In order to realize object of the present invention, in an aspect, the invention provides a kind of bacterial vaginitis LAMP detection kit, it comprises the primer, reaction buffer, BstDNA polysaccharase and the nucleic acid dye that design for gardnerella vaginalis conservative region, and the nucleotide sequence of wherein said primer is as follows:
Outer primer F3:ATAGCTCTTGGAAACGGGTG (SEQIDNO:1)
Outer primer B3:CGCAATATTCCCCACTGCTG (SEQIDNO:2)
Inner primer FIP:ACCCCATCCCATGCCACTAAAC-AATGCTGGATGCTCCAACTT (SEQIDNO:3)
Inner primer BIP:ACCTAGGCTTCGACGGGTAGC-TGGGCCGTATCTCAGTCC (SEQIDNO:4).
Preferably, the concentration of described outer primer F3 and described outer primer B3 is 5pmol/ μ l, and the concentration of described inner primer FIP and described inner primer BIP is 40pmol/ μ l.
Preferably, described reaction buffer is by 10mMdNTP, 10 × ThermoPol reaction buffer, 50mMMgSO
4form with 5mM trimethyl-glycine.
Preferably, the concentration of described BstDNA polysaccharase is 8U/ μ l.
Preferably, described nucleic acid dye is 1000 × SYBRGreenI.
Preferably, test kit of the present invention comprises DNA extraction reagent and positive control and negative control further.
Preferably, described DNA extraction reagent is CTAB extraction buffer, and it is made up of 100mMTris-HClpH8.0,50mMEDTA, 1MNaCl, 1% (v/v) beta-mercaptoethanol, 2%CTAB, pH7.0-7.5.
In one aspect of the method, the present invention also provides a kind of bacterial vaginitis LAMP detection method, and it comprises the following steps:
(1) extract testing sample DNA: in testing sample, add CTAB extraction buffer, conveniently CTAB method extracts DNA;
(2) LAMP reaction system is set up: in PCR pipe, prepare 25 μ l reaction systems, it comprises the outer primer F31 μ l of 5pmol/ μ l, the outer primer B31 μ l of 5pmol/ μ l, the inner primer FIP1 μ l of 40pmol/ μ l, inner primer BIP1 μ l, the reaction buffer 2.5 μ l of 40pmol/ μ l, the BstDNA polysaccharase 1 μ l of 8U/ μ l, template DNA 2 μ l, add water and be supplemented to 25 μ l, and using gardnerella vaginalis genomic dna as positive control, using 100mMTris-HClpH8.0 and 50mMEDTA as negative control;
(3) LAMP reaction: by the PCR pipe in step (2) in 63 DEG C of isothermal reaction 60min;
(4) analysis judges reaction product result: in (3), add 1 μ l nucleic acid dye in gained reaction product, and if reaction solution color is orange, represent that result is negative, if reaction solution color is green, expression result is positive.
In one aspect of the method, present invention also offers primer and preparing the application in bacterial vaginitis LAMP kit, wherein said primer is for the design of gardnerella vaginalis conservative region, and nucleotide sequence is as follows:
Outer primer F3:ATAGCTCTTGGAAACGGGTG (SEQIDNO:1)
Outer primer B3:CGCAATATTCCCCACTGCTG (SEQIDNO:2)
Inner primer FIP:ACCCCATCCCATGCCACTAAAC-AATGCTGGATGCTCCAACTT (SEQIDNO:3)
Inner primer BIP:ACCTAGGCTTCGACGGGTAGC-TGGGCCGTATCTCAGTCC (SEQIDNO:4).
Bacterial vaginitis LAMP detection kit of the present invention and detection method have following beneficial effect:
1, high specific: according to conserved regions design four Auele Specific Primers of gardnerella vaginalis gene, by the existence of loop-mediated isothermal amplification reaction and judgement target substance whether, false positive rate is low;
2, consuming time short: increasing can complete in 30-60min, fast, efficient and productive rate is high;
3, highly sensitive: the target that individual cells can be detected in complex system, the lowest detection limit reaches 1pgDNA, and the recall rate of sample reaches more than 98%;
4, identify easy: by visual color change get final product result of determination, without the need to high-end plant and instrument and the step such as cumbersome electrophoresis and ultraviolet visualization.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
The preparation of embodiment 1 bacterial vaginitis LAMP detection kit of the present invention
1.1 reagent
Primer is synthesized by TIANGEN Biotech (Beijing) Co., Ltd.; BstDNA polysaccharase and 10 × ThermoPol reaction buffer are purchased from NEB; SYBRGreenI is purchased from Invitrogen; All the other PCR reagent and the reagent needed for preparation CTAB extraction buffer are purchased from Sigma.
The preparation of 1.2 test kits:
Obtain following reagent, to prepare bacterial vaginitis LAMP detection kit of the present invention:
CTAB extraction buffer: according to following formulated: 100mMTris-HClpH8.0,50mMEDTA, 1MNaCl, 1% (v/v) beta-mercaptoethanol, 2%CTAB, adjusted to ph is to 7.0-7.5;
Reaction buffer: according to following formulated: 10mMdNTP, 10 × ThermoPol reaction buffer, 50mMMgSO
4, 5mM trimethyl-glycine;
Primer: outer primer F3, its nucleotide sequence is as shown in SEQIDNO:l; Outer primer B3, its nucleotide sequence is as shown in SEQIDNO:2; Inner primer FIP, its nucleotide sequence is as shown in SEQIDNO:3, and inner primer BIP, its nucleotide sequence is as shown in SEQIDNO:4.Wherein the concentration of outer primer F3 and outer primer B3 is 5pmol/ μ l, and the concentration of inner primer FIP and inner primer BIP is 40pmol/ μ l.
Concentration is the BstDNA polysaccharase of 8U/ μ l;
Nucleic acid dye: 1000 × SYBRGreenI;
Positive control: gardnerella vaginalis genomic dna;
Negative control: 100mMTris-HCl (pH8.0) and 50mMEDTA.
Embodiment 2 gardnerella vaginalis specific detection
2.1LAMP specific detection
Test strains comprises the gardnerella vaginalis being separated from BV patient's vaginal secretions and obtaining, and each 1 strain of peptostreptococcus, purple Zymomonas mobilis and influenzae.
The test kit using embodiment 1 to prepare detects gardnerella vaginalis according to following steps:
(1) extract testing sample DNA: be inoculated in by test strains on PDA flat board, after streak culture, collection is also centrifugal, adds 50 μ lCTAB extraction buffers, and conveniently CTAB method extracts DNA;
(2) LAMP reaction system is set up: in PCR pipe, prepare 25 μ l reaction systems, wherein four kinds of each 1 μ l of primer, reaction buffer 2.5 μ l, the BstDNA polysaccharase 1 μ l of 8U/ μ l, step (1) gained template DNA 2 μ l, add water and be supplemented to 25 μ l, and using gardnerella vaginalis genomic dna as positive control, using 100mMTris-HClpH8.0 and 50mMEDTA as negative control;
(3) LAMP reaction: by PCR pipe in step (2) in 63 DEG C of isothermal reaction 60min;
(4) analysis judges reaction product result: in (3), add 1 μ l1000 × SYBRGreenI in gained reaction product, and if reaction solution color is orange, represent that result is negative, if reaction solution color is green, expression result is positive.
2.2 detected result
All present green in the PCR pipe of gardnerella vaginalis, and its colour developing result making bacterial strain is orange, shows that primer has very strong specificity.
Embodiment 3 gardnerella vaginalis sensitivity technique
3.1LAMP sensitivity technique
Extract gardnerella vaginalis DNA according to the step (1) of embodiment 2, and be diluted to 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg totally 7 gradients with 10 times of concentration series dilution methods.
LAMP reaction system and reaction conditions and interpretation of result are with step (2)-(4) of embodiment 2.
3.2 detected result
Except DNA concentration be 100fg PCR pipe display orange except, all present green in the PCR pipe of all the other concentration, show that the lowest detection limit of detection method of the present invention reaches 1pgDNA, sensitivity is very high.
Claims (5)
1. a bacterial vaginitis LAMP detection kit, is characterized in that, can comprise primer, reaction buffer, BstDNA polysaccharase and nucleic acid dye, the nucleotide sequence of wherein said primer is as follows:
Outer primer F3:ATAGCTCTTGGAAACGGGTG (SEQIDNO:1)
Outer primer B3:CGCAATATTCCCCACTGCTG (SEQIDNO:2)
Inner primer FIP:ACCCCATCCCATGCCACTAAAC-AATGCTGGATGCTCCAACTT (SEQIDNO:3)
Inner primer BIP:ACCTAGGCTTCGACGGGTAGC-TGGGCCGTATCTCAGTCC (SEQIDNO:4).
2. bacterial vaginitis LAMP detection kit according to claim 1, wherein said reaction buffer is by 10mMdNTP, 10 × ThermoPol reaction buffer, 50mMMgSO
4form with 5mM trimethyl-glycine.
3. bacterial vaginitis LAMP detection kit according to claim 1, wherein said nucleic acid dye is 1000 × SYBRGreenI.
4. the bacterial vaginitis LAMP detection kit according to any one of claim 1-3, it comprises DNA extraction reagent and positive control and negative control further.
5. primer is preparing the application in bacterial vaginitis LAMP detection kit, and the nucleotide sequence of wherein said primer is as follows:
Outer primer F3:ATAGCTCTTGGAAACGGGTG (SEQIDNO:1)
Outer primer B3:CGCAATATTCCCCACTGCTG (SEQIDNO:2)
Inner primer FIP:ACCCCATCCCATGCCACTAAAC-AATGCTGGATGCTCCAACTT (SEQIDNO:3)
Inner primer BIP:ACCTAGGCTTCGACGGGTAGC-TGGGCCGTATCTCAGTCC (SEQIDNO:4).
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| CN201610017748.1A CN106350581B (en) | 2015-07-13 | 2015-07-13 | Method for detecting bacterial vaginitis by using detection kit |
| CN201510409316.0A CN104911275B (en) | 2015-07-13 | 2015-07-13 | A kind of bacterial vaginitis detection kit |
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| CN105420360A (en) * | 2015-12-09 | 2016-03-23 | 广州和实生物技术有限公司 | Kit for instantly detecting vaginitis pathogen gene |
| EP3714061A4 (en) * | 2017-11-24 | 2021-08-11 | The University Of Western Australia | Infection-related preterm birth diagnostic method |
| AU2019324196A1 (en) | 2018-08-24 | 2021-03-18 | Gen-Probe Incorporated | Compositions and methods for detecting bacterial nucleic acid and diagnosing bacterial vaginosis |
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|---|---|
| CN105087812B (en) | 2016-10-12 |
| CN105087812A (en) | 2015-11-25 |
| CN104911275A (en) | 2015-09-16 |
| CN106350581B (en) | 2020-09-18 |
| CN106350581A (en) | 2017-01-25 |
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