CN110343775A - The primer of double check Gardner bacillus and trichomonas vaginalis, probe groups, kit and detection method - Google Patents

The primer of double check Gardner bacillus and trichomonas vaginalis, probe groups, kit and detection method Download PDF

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CN110343775A
CN110343775A CN201910631817.1A CN201910631817A CN110343775A CN 110343775 A CN110343775 A CN 110343775A CN 201910631817 A CN201910631817 A CN 201910631817A CN 110343775 A CN110343775 A CN 110343775A
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probe
seq
nucleotide sequence
kit
primer
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蔺皓
郭光华
邹国宝
魏颖颖
宋高尚
吴茜
刘欣欣
沈江卫
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China Biotechnology International Ltd By Share Ltd
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    • 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]
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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
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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
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
    • 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

Abstract

The present invention relates to the detection technique field of pathogenic microorganism more particularly to primer, probe groups, kit and the detection methods of double check Gardner bacillus and trichomonas vaginalis.It include GD/TV conservative region special primer pair and Taqman fluorescence probe in the kit, the kit passes through real-time fluorescence quantitative PCR, accurately GD/TV infection pattern detection can be come out, high sensitivity, specificity are good, repeated strong and convenient to use.

Description

The primer of double check Gardner bacillus and trichomonas vaginalis, probe groups, kit and Detection method
Technical field
The present invention relates to the detection technique field of pathogenic microorganism more particularly to double check Gardner bacillus and vagina hairs Primer, probe groups, kit and the detection method of trichomonad.
Background technique
Vaginitis (vaginitis) i.e. colpitis is to lead to vulvovaginal symptom such as itch, cusalgia, stimulation and exception One group of illness of flow liquid.There are aerobic bacteria and anaerobic bacteria to reside in intravaginal under normal circumstances, forms normal vaginal flora.It is any Reason breaks the ecological balance between vagina and flora, can also formation condition pathogenic bacteria.It is clinically common to have: bacterial vaginosis Sick (occupying symptom women 22%~50%), monilial vaginitis (17%~39%), trichomonas vaginitis (4%~ 35%), senile vahinitis, infantile vaginitis.The pathogen of vaginitis is effectively identified and diagnosed, for vaginitis Symptomatic treatment it is most important.
The main pathogen of bacterial vaginosis BV (Bacterial vaginosis, BV) is vagina Gardner bacillus (Gardnerella vaginalis, GD), can be through transmission through sex.Gardner bacillus is the tiny bacillus of Gram-negative, Chang Cheng Club-shaped, sometimes in Filamentous and Multiple Shape, common bipolar staining.It is 0.4--0.6 microns wide, long 1--2 microns of size, non-activity Power, atrichia, without gemma.Many bacterial strains have pod membrane.Infection caused by Gardner bacillus is most lighter, and the property be more common in enlivens woman Female.Acute stage leucorrhea increasing has the stink of fish raw meat or ammonia, and vulva is moist uncomfortable, is often accompanied by vagina burning heat sensation, intercourse pain and vulva It disturbs and itches.Inspection is shown in that some patientss vulva is red and swollen, and vagina mucosa is congested, is in bois de rose, and mild redness, secretion is in homogenieity more, Thin, canescence is sometimes milk yellow or class green, bad smell.Vagina pH is often 5--5.5.Sometimes leukorrhea amount is few, only thin It is one layer thin, it is covered on such as film sample on congested vaginal wall.Small number of patients vaginal wall has erythema or petechiae, and pregnant patient can cause to flow Production or puerperal endometritis.Infection severe one may also lead to septicemia, urinary tract infection, perinephric abscess and cystitis etc..
Trichomonas vaginitis is common gynecological disease, is caused by trichomonas vaginalis (Trichomonas vaginalis).Hair drop Worm is in wide pyriform or ellipse, and a length of 10~30 μm, 10~20 μm wide, there are 4 flagellums isometric with polypide on head, in microscope Down it will be clear that this trichmonad.Trichmonad is very strong to different environment-adapting abilities, can be under the conditions of 25 DEG C~42 DEG C Growth and breeding, 3 DEG C~5 DEG C of low temperature can survive 21 days, remain to existence 20~60 minutes at 46 DEG C, be detached from after human body half-dried It can also survive a few hours under conditions of dry.Duration vaginitis is shown as after infection in women, onset can suddenly be delayed.Clinical manifestation is Vaginal fluid increases, show bubble, bad smell taste, yellow green.Dysuria, pruritus vulvue.Acute stage is for 1 week or the several months, State of an illness weight has fluctuation, dyspareunia, exacerbation of symptoms after the menstrual period.Subsequent leukorrhea is reduced, and symptom mitigates, and can also be completely disappeared. If trichomonas vaginalis is parasitic in urethra or bladder, trichmonad property urethra, cystitis can be caused.Trichomonas vaginalis can swallow essence Son can cause infertile.Trichomonas vaginalis can also cause cell development exception and dyskaryosis, and the incidence of cancer is significantly higher than nothing Trichomonad women.Discovery is checked, from fornix vagina and cervix mild hyperaemia to extensive rotten to the corn, petechiae and crissum be rotten to the corn and flush Endometrium (strawberry shape cervix).
Currently, the detection method of Gardner bacillus (GD), trichomonas vaginalis (Tv) property vaginitis (referred to as GD/Tv) is main It is separately cultured, vaginitis five/seven and diagnosis of molecular biology, three kinds of methods have the following characteristics that
1) pathogen be separately cultured be GD/Tv goldstandard, but isolated culture is higher to technical requirements, and expense is relatively high Expensive, time-consuming, using few in clinical detection.
2) vaginitis five-seven are current clinical detections using more laboratory method, predominantly detect vaginitis and draw The index of correlation variation risen, including catalase, neuraminidase, leukocyte esterase, Prolyl iminopeptidase, acetylamino Glucuroide, oxidizing ferment, pH value.But these indexs are indirect detection, have inexactness.
3) pathogen nucleic acid detects: have many advantages, such as that quick, accurate, detection window is short, highly sensitive, it can be to GD/Tv Early diagnosis is made, provides strong technical support to the quick analysis and control of the state of an illness.
Wherein real-time quantitative PCR detection platform has become most simple and direct and reliable nucleic acid detection method, domestic and international at present Diagnostic nucleic acid in be most widely used, testing principle is, detection probe be include 5 ' end reporter groups and 3 ' end quenching groups Oligonucleotides, when probe is complete, due to quenching group close to reporter group greatly reduce reporter group sending it is glimmering Light, when primer extend, the probe in conjunction with template is cut off by Taq enzyme (5 ' → 3 ' exonuclease activity), reporter group with quench The group that goes out separation, generates fluorescence signal.In each PCR cycle, there is new reporter group to be sheared, therefore fluorescence signal is strong The increase of degree and the quantity of amplified production are directly proportional, can monitor amplification situation in real time.
Currently, domestic be directed to vagina hair road trichomonad, there are the patent individually detected less than ten, Publication No. The patent of CN102719529B discloses trichomonas vaginalis and Candida albicans binary channels fluorescence PCR detecting method and its reagent Box is two re-detections for trichomonas vaginalis and Candida albicans；And it is directed to the fluorescence quantitative PCR detection pole of Gardner bacillus Few, more without being directed to GD/Tv joint-detection PCR kit for fluorescence quantitative product.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is that providing double check Gardner bacillus and trichomonas vaginalis Primer, probe groups, kit and the detection method of Multiple detection, the targeted target fragment of the primer, probe groups is smaller, special It is anisotropic stronger, and sensitivity is higher.
Primer, the probe groups of Gardner bacillus provided by the invention and trichomonas vaginalis Multiple detection comprising:
The primer pair of nucleotide sequence shown in NO:1~2 SEQ ID, the probe of nucleotide sequence shown in SEQ ID NO:3；
The primer pair of nucleotide sequence shown in NO:4~5 SEQ ID, the probe of nucleotide sequence shown in SEQ ID NO:6.
The present invention selects Gardner bacillus 16SrRNA conservative region, trichomonas vaginalis ITS conservative region, beta-globin base Because of (HBB, internal standard) conservative region, specific primer and probe are designed, using Real-Time Fluorescent Quantitative PCR Technique, GD/Tv reality is made When fluorescent quantificationally PCR detecting kit, and describe its application method.
In the present invention, the primer pair of nucleotide sequence shown in NO:1~2 SEQ ID, nucleotides sequence shown in SEQ ID NO:3 The probe of column is designed for the 16S sRNA of Gardner bacillus (GD), and the GD specific target gene fragment length is 127bp；Sequence It is classified as: CGGATCGTAGTCTGCAACTCGACTACGTGAAGGCGGAGTCGCTAGTAATCGCGAAT CAGCAACGTCGCGGT GAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCA(SEQ ID NO:10)。
In the present invention, the primer pair of nucleotide sequence shown in NO:4~5 SEQ ID, nucleotides sequence shown in SEQ ID NO:6 The probe of column is designed for the ITS of trichomonas vaginalis (Tv), and the Tv specific target gene fragment length is 153bp, sequence are as follows: CAGGTTAATCTTTGAATGCAAATTGCGCTAAACTCGATCTCGGTCGAGAAGCATGGGTGTGACAGTACTACATCTT TTATAATAATTCTTATTCTAAGCGAATAAGTAAATAATATATAAGACAAACAACACGTAGTCTGCCATACGCAGGAA (SEQ ID No.11)。
Primer and probe of the present invention has good accuracy, sensitivity, specificity, even if in the same amplification It is detected in system, it also being capable of good differentiation GD or Tv.This kit to the minimum detectability concentration of GD or Tv be 1.0 × 100Copy/μ L, i.e. minimum detectability are 5 copies.
In the present invention, 5 ' ends of the probe are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching group.
Specifically, 5 ' ends of the probe of nucleotide sequence shown in SEQ ID NO:3 are marked with fluorescent reporter group CY5,3 ' End is marked with fluorescent quenching group BHQ3；
5 ' ends of the probe of nucleotide sequence shown in SEQ ID NO:6 are marked with fluorescent reporter group FAM, and 3 ' ends are marked with Fluorescent quenching group MGB.
The present invention also provides the kits of Gardner bacillus and trichomonas vaginalis Multiple detection comprising of the present invention Primer, probe groups.
It further include the primer pair and probe to reference gene HBB detection in kit provided by the invention；
The nucleotide sequence of the primer pair for reference gene HBB detection is as shown in NO:7~8 SEQ ID；It is described For reference gene HBB detection probe nucleotide sequence as shown in SEQ ID NO:9.The HBB specific target gene piece Duan Xulie are as follows: TCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGG ATGAAGTTGGT GGTGAGGCCCTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATG(SEQ ID NO:12).In kit provided by the invention, 5 ' ends of the probe of nucleotide sequence shown in SEQ ID NO:12 are marked with glimmering Light reporter group ROX, 3 ' ends are marked with fluorescent quenching group BHQ2.
It further include fluorescence quantitative PCR detection reagent and/or sample extraction reagent in kit provided by the invention；
The fluorescence quantitative PCR detection reagent includes qPCR Master Mix enzyme mixation, qPCR Master Mix anti- Answer buffer；
The qPCR Master Mix enzyme mixation, including Taq enzyme and UNG enzyme, the Taq enzyme are AnstartTaq Archaeal dna polymerase, the UNG enzyme are uracil-N-glycosylase.
The sample extraction reagent includes NaOH, TritonX-100 and TE buffer.
The sample extraction liquid includes NaOH final concentration of 0.01%~0.5%, and preferably 0.1%.
The sample extraction liquid includes TritonX-100 final concentration of 0.01%~0.5%, and preferably 0.1%.
It further include positive control and/or negative control in kit provided by the invention；
Negative controls of the invention are DEPC water；
Positive reference substance of the invention is the plasmid solution containing Gardner bacillus and trichomonas vaginalis target fragment.
The present invention also provides the methods for detecting and distinguishing Gardner bacillus and trichomonas vaginalis comprising: with the present invention The kit carries out real-time fluorescence quantitative PCR detection to sample to be tested DNA, and it is then positive for generating S type amplification curve.
In method of the present invention, the system of the real-time fluorescence quantitative PCR includes:
Water complements to 25 μ L.
In system, the primer concentration is 100~1000nM, preferably 500nM.The concentration and probe concentration is 10~500nM, Preferably 200nM.
In method of the present invention, the program of the real-time fluorescence quantitative PCR includes:
50 DEG C 2 minutes；
95 DEG C of 5 minutes initial denaturations；
95 DEG C 15 seconds → 60 DEG C 35 seconds, 45 circulation.
Judgement for result, specifically includes:
Value < 38 amplification curve Ct of measuring samples DNA and there is typical S type amplification curve, is positive findings；
The amplification curve Ct value of measuring samples DNA is equal to 45 (or being shown as Undet) or without typical S type amplification curve, is Negative findings；
Value < 45 38≤Ct of amplification curve of DNA sample to be checked are gray area, and such as resurveying result still is value < 45 Ct, and There is typical S type amplification curve, is then determined as positive findings；As Ct value is not shown still or without typical S type amplification curve, is then determined as Negative findings.
In the case that measuring samples GD or Tv are feminine gender, investigating HBB amplification situation or HBB does not have S type amplification curve, Or Ct value is greater than 35, shows sampling failure, extracts failure or sample-adding mistake, need to test again.
For the experiment of positive control is arranged, should also meet:, there is apparent expansion in positive control, the channel CY5 at the channel FAM Increase, there is typical case's S type amplification curve, and Ct value is less than 30；
For be arranged negative control experiment, should also meet: negative control, the channel CY5, the channel FAM, value=45 Ct (or It is shown as undet) or without typical S type amplification curve, subsequent judgement is carried out, it otherwise should carry out trouble-shoots.
Primer and probe of the present invention can be used for the detection of a variety of source samples.The detection of biological sample can be sentenced Whether the sample break by GD or Tv infection, and may determine that the sample whether by GD or Tv pollution the detection of non-biological specimen. Therefore, kit of the present invention can be used for the detection of the non-diagnostic purpose of sample.In the present invention, the sample of the detection comes From vagina, medical instrument, drug, food or cosmetics.In some specific embodiments, the sample to be tested vaginal fluid.
Beneficial effects of the present invention include at least:
(1) invention is directed to the conservative region of GD/Tv/HBB, is total to specific designs 6 primers and 3 Taqman probes, In conjunction with the conservative region of target gene, there is very high specificity, with pseudomonas aeruginosa strains CMCC10104, golden yellow Aureus strains CMCC26001, Neisseria meningitidis strain CMCC29108, lactobacterium casei strains CMCC34103, large intestine angstrom are uncommon Salmonella strain CMCC44103, deformed rod bacterial strain CMCC49102, bacterium typhosum strain CMCC50096, shigella flexneri strain CMCC51573, Candida albicans bacterial strain CMCC98001, mycoplasma hominis (MH), herpes simplex virus (HSV), human papilloma virus (HPV), human cytomegalovirus (HCMV), mycoplasma pneumoniae (MP), mycoplasma genitalium (MG), chlamydia trachomatis (CT), gonococcus (NG), urea substance (UU) is without cross reaction；
(2) detection of 2 kinds of pathogens of GD/Tv is completed in same pipe, and is subject to area by different fluorescence labeling probes Point, it can clinically be extracted by a sample nucleic acid, a nucleic acid sample-adding, complete the synchronous detection of 2 kinds of pathogens, be effectively reduced Previous single tube detects the excessively high problem of bring testing cost, and is effectively improved the cumbersome situation of single tube detection；
(3) target gene segment is shorter, between 100~160bp, if the too short effect of depolluting that will affect UNG enzyme, If too long will cause reaction efficiency decline, therefore the present invention can effectively avoid pollution and the high efficiency and spirit of reaction Sensitivity can detect the GD/Tv DNA copied down to 5；
(4) human p-globin's gene-specific primer probe is added in reaction tube, it can effective monitoring specimen sample, sample This extraction, PCR detect overall process, overcome and external standard is added in extraction process, be unable to the disadvantage of effective monitoring specimen sample success or not End.
Kit of the invention is very suitable to clinical use and popularization, the prevention and control to GD/Tv in the women vaginitis in China Work has great importance.
Detailed description of the invention
Fig. 1 is the fluorescence detection of 4 specificity experiments of embodiment as a result, the corresponding template of each detection curve is 1: verdigris is false single Born of the same parents' bacteria strain CMCC10104,2: staphylococcus aureus strains CMCC26001,3: Neisseria meningitidis strain CMCC29108,4: Lactobacterium casei strains CMCC34103,5: escherichia coli bacterial strain CMCC44103,6: deformed rod bacterial strain CMCC49102,7: typhoid fever Salmonella strain CMCC50096,8: shigella flexneri strain CMCC51573,9: Candida albicans bacterial strain CMCC98001,10: human-like Mycoplasma (MH), 11: herpes simplex virus (HSV), 12: human papilloma virus (HPV), 13: human cytomegalovirus (HCMV), 14: mycoplasma pneumoniae (MP), 15: mycoplasma genitalium (MG), 16: chlamydia trachomatis (CT), 17: gonococcus (NG), 18: urea substance (UU) and 19: positive reference substance；
Fig. 2 is GD (channel the Cy5) fluorescence detection of 5 sensitivity experiment of embodiment as a result, the corresponding template of each detection curve It is 1~7 for the plasmid solution containing GD/Tv/HBB target fragment, concentration is followed successively by 1.0 × 106、1.0×105、1.0× 104、1.0×103、1.0×102、1.0×101、1.0×100Copy/μ L；
Fig. 3 is Tv (channel the FAM) fluorescence detection of 5 sensitivity experiment of embodiment as a result, the corresponding template of each detection curve It is 1~7 for the plasmid solution containing GD/Tv/HBB target fragment, concentration is followed successively by 1.0 × 106、1.0×105、1.0× 104、1.0×103、1.0×102、1.0×101、1.0×100Copy/μ L；
Fig. 4 is the fluorescence detection result of 100 times of positive control dilutions product (R1) of 6 repetitive test of embodiment.
Specific embodiment
The present invention provides the primer of Gardner bacillus and trichomonas vaginalis double check, probe, kit and detection sides Method, those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that all Similar replacement and change is apparent to those skilled in the art, they are considered as being included in the present invention.This The method of invention and application are described by preferred embodiment, and related personnel can obviously not depart from the present invention Hold, in spirit and scope to methods herein and application is modified or appropriate changes and combinations, carrys out the implementation and application present invention Technology.
The test material that the present invention uses is all common commercially available product, can all be bought in market.
Below with reference to embodiment, the present invention is further explained:
Embodiment 1: the present invention quickly detects the design of the primed probe pair of GD/Tv
Gardner bacillus 16SrRNA sequence, trichomonas vaginalis ITS sequence, HBB gene sequence in NCBI are downloaded, design is drawn Object to and probe, sequence it is as follows:
The primer and probe sequence of the present invention of table 1
Embodiment 2: for quickly detecting the foundation of the real-time fluorescence quantitative PCR kit of GD/Tv
For quickly detecting the real-time fluorescence quantitative PCR kit of people GD/Tv, including reaction mixture, sample extraction liquid, Positive reference substance, negative controls, specification and box body.
Wherein reaction mixture contains upstream and downstream primer and probe (NO:1~12 SEQ ID), Anstart qPCR Master Mix enzyme mixation, Anstart qPCR Master Mix5 × reaction buffer.
Wherein Anstart qPCR Master Mix enzyme mixation, Anstart qPCR Master Mix5 × reaction are slow Fliud flushing is provided by Fei Peng Biological Co., Ltd., and Anstart qPCR Master Mix enzyme mixation dilutes 25 times of uses, Anstart qPCR Master Mix5 × reaction buffer dilutes 5 times of uses.
Wherein primer GD-F, GD-R, Tv-F, Tv-R, HBB-F, HBB-R final concentration 500nM.
Its middle probe GD-P, Tv-P, HBB-R concentration is 200nM.
Wherein GD-P fluorescence probe group is Cy5, and quencher BHQ3, Tv-P fluorescence probe group is FAM, quenches base Group is MGB, and HBB-P fluorescence probe group is ROX, quencher BHQ2.
Wherein sample extraction liquid is the TE buffer containing 0.1%NaOH, 0.1%TritonX-100.
Wherein positive reference substance, for the plasmid solution containing GD/Tv/HBB target fragment, concentration is 10000 copies/μ L, corresponding Ct value is 23 or so.
Wherein GD specific target gene fragment sequence are as follows:
CGGATCGTAGTCTGCAACTCGACTACGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGC GGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCA(SEQ ID No.10)。
Wherein Tv specific target gene fragment sequence are as follows:
CAGGTTAATCTTTGAATGCAAATTGCGCTAAACTCGATCTCGGTCGAGAAGCATGGGTGTGACAGTAC TACATCTTTTATAATAATTCTTATTCTAAGCGAATAAGTAAATAATATATAAGACAAACAACACGTAGTCTGCCAT ACGCAGGAA(SEQ ID No.11)。
Wherein HBB specific target gene fragment sequence are as follows:
TCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTG GTGAGGCCCTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATG(SEQ ID No.12)。
Wherein negative controls are DEPC water.
The rapid detection method of embodiment 3:GD/Tv kit for detecting nucleic acid
The GD/Tv in people's vaginal fluid is quickly detected using the kit of embodiment 2, the specific steps are as follows:
(1) it nucleic acid extraction: (determines whether to be felt by GD or Tv using cultivation to vaginal fluid (10 parts, number 1-10) Dye, this method are current industry goldstandard) 1mL physiological saline, sufficiently oscillation washing cotton swab are added in collecting pipe, then by cotton Swab extracts discarding by wall, and 500 μ L liquid is taken to be transferred in the centrifuge tube of 1.5mL, and 13000rpm is centrifuged 5 minutes, abandons supernatant, sinks 1mL physiological saline is added in shallow lake, breaks up precipitating, 13000rpm is centrifuged 5 minutes, is abandoned supernatant, is added in precipitating and has vibrated mixing 50 μ L of sample extraction liquid is vibrated in vortex oscillator and is broken up precipitating (gently breaing up precipitating with pipette tips when necessary), 100 DEG C of dry baths or Person's water-bath 10 minutes, 13000rpm was centrifuged 5 minutes, and supernatant is for PCR reaction (without the precipitating of touch tube bottom when absorption Object).
(2) real-time quantitative fluorescence PCR: PCR amplification is carried out using above-mentioned GD/Tv real-time fluorescence quantitative PCR detection kit Reaction reacts mixing with kit respectively using the measuring samples DNA of extraction, positive control and each 5 μ L of negative control as template 20 μ L of liquid mixing carries out real-time fluorescence quantitative PCR reaction, the setting of PCR reaction condition are as follows: and 50 DEG C of 2 minutes UNG enzymes depollute, and 95 DEG C 5 minutes initial denaturations, 95 DEG C 15 seconds → 60 DEG C 35 seconds, the channel Cy5, FAM, ROX is for collecting detection fluorescence signal, and totally 45 are followed Ring.
(3) result judges:
1. should meet: there is apparent amplification in positive control, the channel CY5, the channel FAM, have typical case's S type amplification curve, And Ct value is less than 30；Negative control, the channel CY5, value=45 FAM channel C t (or being shown as undet) or bent without the amplification of typical S type Line carries out subsequent judgement, otherwise should carry out trouble-shoots；
It is positive findings 2. value < 38 amplification curve Ct of measuring samples DNA and having typical S type amplification curve；
3. the amplification curve Ct value of measuring samples DNA is equal to 45 (or being shown as Undet) or without typical S type amplification curve, For negative findings；
4. value < 45 38≤Ct of amplification curve of DNA sample to be checked are gray area, such as resurveying result still is value < 45 Ct, And have typical S type amplification curve, then it is determined as positive findings；As Ct value is not shown still or without typical S type amplification curve, is then determined For negative findings；
In the case that 5. measuring samples GD, Tv are feminine gender, investigating HBB amplification situation or HBB does not have S type amplification curve, Or Ct value is greater than 35, shows sampling failure, extracts failure or sample-adding mistake, need to test again.
Determine result such as table 2:
2 sample detection result of table
The results show that 9 parts of GD positive sample, 1 part of Tv positive sample, wherein 1 part of full positive sample.As a result with the prior art Identification is consistent, and accuracy rate is up to 100%.
Embodiment 4: specificity experiments
Using the method for kit, embodiment 3 in embodiment 2 respectively to pseudomonas aeruginosa strains CMCC10104, gold Staphylococcus aureus bacterial strain CMCC26001, Neisseria meningitidis strain CMCC29108, lactobacterium casei strains CMCC34103, large intestine Escherichia strain CMCC44103, deformed rod bacterial strain CMCC49102, bacterium typhosum strain CMCC50096, shigella flexneri Strain CMCC51573, Candida albicans bacterial strain CMCC98001, mycoplasma hominis (MH), herpes simplex virus (HSV), human papilloma virus Malicious (HPV), human cytomegalovirus (HCMV), mycoplasma pneumoniae (MP), mycoplasma genitalium (MG), chlamydia trachomatis (CT), leaching ball Bacterium (NG), urea substance (UU) and positive reference substance carry out PCR detection, the result is shown in Figure 1, the results showed that, contain only GD/Tv target gene piece The plasmid solution of section, i.e. positive reference substance are the positive, remaining pipe is feminine gender.The result shows that detection kit of the invention is special It is anisotropic high, accurately, specifically GD/Tv can detected.
Embodiment 5: sensitivity experiment
Positive reference substance (i.e. the plasmid solution of the target fragment containing GD/Tv/HBB) is taken, its concentration is measured and calculates copy Number is diluted according to 10 times of concentration gradients, chooses 1.0 × 100~1.0 × 106Copy/μ L concentration is as sample, with the present invention Kit and detection method detected.
Testing result is as shown in Fig. 2-Fig. 3, the results showed that, this kit is 1.0 × 10 to the minimum detectability concentration of GD0 Copy/μ L (is equivalent to 100 bacterium/mL of concentration), i.e., minimum detectability is 5 copies.To the minimum detectability concentration of Tv be 1.0 × 100Copy/μ L (is equivalent to 100/mL of concentration), i.e., minimum detectability is 5 copies.
Embodiment 6: repetitive test
100 times of dilution product for taking positive control in kit, are named as R1, with kit and detection method of the invention, 10 tests are continuously repeated, testing result is shown in Fig. 4, table 3, and the 100 of positive control times dilute the product GD value coefficient of variation as the result is shown Respectively less than 5%.
The repeated reference plate Ct value of table 3 and the coefficient of variation (CV value).
Template GD(CY5) Tv(ROX)
R1 31.79 32.18
R1 31.86 32.23
R1 31.86 31.98
R1 31.86 32.00
R1 31.59 32.08
R1 31.72 32.13
R1 31.95 32.44
R1 31.74 31.85
R1 31.92 32.10
R1 31.65 31.67
Average value 31.79 32.06
Standard deviation 0.12 0.21
CV value 0.37% 0.66%
The above result shows that kit of the invention has good accuracy, high sensitivity, repeatability strong, easy to use Feature is suitble to clinical use.
Comparative example 1
4 primer and probe sequence of table
Positive reference substance (i.e. the plasmid solution of the target fragment containing GD/Tv) is taken, its concentration is measured and calculates copy number, press According to 10 times of concentration gradient dilution, 1.0 × 10 are chosen0~1.0 × 106Copy/μ L concentration is as sample, described in comparative example 1 Primer and probe detected.
The result shows that the primer and probe of comparative example 1 is that 100 copies/μ L (is equivalent to dense to the minimum detectability concentration of GD Spend 10000 bacterium/mL), i.e., minimum detectability is 500 copies.Minimum detectability concentration to Tv is that 100 copies/μ L (is equivalent to 10000/mL of concentration), i.e., minimum detectability is 50 copies.The results show that the primer and probe reaction efficiency of comparative example 1 is inclined Low, sensitivity is insufficient.
Embodiment 7: Amino-Cerv influences pattern detection
Selection common Amino-Cerv and cleaning product on the market, including anti-cervical erosion suppository, Nifuratel nystatin expandable vaginal soft capsule, It is belle's health carbomer gel, the clean carbomer gel of cervicitis, ofloxacin gel, ofloxacin gel, metronidazole furanone bolt, clean The negative washing lotion of that, is tested, its influence to kit detection is verified.
The result shows that 0.1mg/ml anti-cervical erosion suppository, 10mg/ml Nifuratel nystatin expandable vaginal soft capsule, 10mg/mL belle's health Carbomer gel, the clean carbomer gel of 10mg/mL cervicitis, 10mg/mL ofloxacin gel, 10mg/mL metronidazole furanone bolt, 0.1% JIEERYIN XIYE can accurately detect to infect in drug with the presence or absence of GD or Tv to detection there is no interference.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.
Sequence table
<110>Zhong Shengfang political affairs Biotechnology Ltd.
<120>primer of double check Gardner bacillus and trichomonas vaginalis, probe groups, kit and detection method
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ttcctgcgta tggcagacta cgtgt 25
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cggatcgtag tctgcaactc gactacgtga aggcggagtc gctagtaatc gcgaatcagc 60
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gacaaacaac acgtagtctg ccatacgcag gaa 153
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tctgactcct gaggagaagt ctgccgttac tgccctgtgg ggcaaggtga acgtggatga 60
agttggtggt gaggccctgg gcaggttggt atcaaggtta caagacaggt ttaaggagac 120
caatagaaac tgggcatg 138

Claims (10)

1. the primer of double check Gardner bacillus and trichomonas vaginalis, probe groups comprising:
The primer pair of nucleotide sequence shown in NO:1~2 SEQ ID, the probe of nucleotide sequence shown in SEQ ID NO:3；With
The primer pair of nucleotide sequence shown in NO:4~5 SEQ ID, the probe of nucleotide sequence shown in SEQ ID NO:6.
2. primer according to claim 1, probe groups, which is characterized in that
5 ' ends of the probe of nucleotide sequence shown in SEQ ID NO:3 are marked with fluorescent reporter group CY5, and 3 ' ends are marked with fluorescence Quenching group BHQ3；
5 ' ends of the probe of nucleotide sequence shown in SEQ ID NO:6 are marked with fluorescent reporter group FAM, and 3 ' ends are marked with fluorescence Quenching group MGB.
3. the kit of double check Gardner bacillus and trichomonas vaginalis comprising primer of any of claims 1 or 2, spy Needle group.
4. kit according to claim 3, which is characterized in that further include to reference gene HBB detection primer pair and Probe；
The nucleotide sequence of the primer pair for reference gene HBB detection is as shown in NO:10~11 SEQ ID；
The nucleotide sequence of the probe for reference gene HBB detection is as shown in SEQ ID NO:12.
5. kit according to claim 4, which is characterized in that the probe of nucleotide sequence shown in SEQ ID NO:12 5 ' ends are marked with fluorescent reporter group ROX, and 3 ' ends are marked with fluorescent quenching group BHQ2.
6. kit according to claim 3, which is characterized in that further include fluorescence quantitative PCR detection reagent and/or sample Extract reagent；
The fluorescence quantitative PCR detection reagent includes that qPCR Master Mix enzyme mixation, qPCR Master Mix reaction are slow Fliud flushing；
The sample extraction reagent includes NaOH, TritonX-100 and TE buffer.
7. kit according to claim 3, which is characterized in that further include positive control and/or negative control；
Negative controls of the invention are DEPC water；
Positive reference substance of the invention is the plasmid solution containing Gardner bacillus and trichomonas vaginalis target fragment.
8. the method for detecting and distinguishing Gardner bacillus and trichomonas vaginalis characterized by comprising
With the described in any item kits of claim 3~7, real-time fluorescence quantitative PCR detection is carried out to sample to be tested DNA, is produced Raw S type amplification curve is then positive.
9. according to the method described in claim 8, it is characterized in that, the system of the real-time fluorescence quantitative PCR includes:
10. according to the method described in claim 8, it is characterized in that, the program of the real-time fluorescence quantitative PCR includes:
50 DEG C 2 minutes；
95 DEG C of 5 minutes initial denaturations；
95 DEG C 15 seconds → 60 DEG C 35 seconds, 45 circulation.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113136438A (en) * 2021-05-14 2021-07-20 广东省农业科学院动物卫生研究所 Nucleic acid molecule, PCR primer pair and kit for detecting avian trichomonas beta-tubulin gene
CN113151308A (en) * 2021-05-10 2021-07-23 广东省农业科学院动物卫生研究所 Nucleic acid molecule, PCR primer pair and kit for detecting avian trichomonas TBP gene
CN116179731A (en) * 2023-03-31 2023-05-30 斯贝福（北京）生物技术有限公司 Primer probe set and kit for detecting trichomonas muriaticus and detection method

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120103801A (en) * 2011-03-11 2012-09-20 (주)팸메드 Methods for simultaneously detecting sexually transmitted disease-inducible microorganisms
CN102719529A (en) * 2012-05-04 2012-10-10 辽宁科骏生物有限公司 Trichomonas vaginalis and candida albicans two-channel fluorescence PCR detection method and kit thereof
US20130288988A1 (en) * 2012-02-20 2013-10-31 Laboratory Corporation Of America Holdings Diagnostic Methods and Markers for Bacterial Vaginosis
CN107099618A (en) * 2017-05-03 2017-08-29 上海速创诊断产品有限公司 A kind of LAMP primer composition thing and its related application for being used to detect urogenital tract pathogenic microorganisms
US20180044739A1 (en) * 2010-01-22 2018-02-15 Gen-Probe Incorporated Oligonucleotides for use in determiniing the presence of trichomonas vaginalis in a sample
CN109487013A (en) * 2019-01-09 2019-03-19 中生方政生物技术股份有限公司 Herpes simplex virus I-type and II type detection marker, primed probe are to, kit and detection method

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180044739A1 (en) * 2010-01-22 2018-02-15 Gen-Probe Incorporated Oligonucleotides for use in determiniing the presence of trichomonas vaginalis in a sample
KR20120103801A (en) * 2011-03-11 2012-09-20 (주)팸메드 Methods for simultaneously detecting sexually transmitted disease-inducible microorganisms
US20130288988A1 (en) * 2012-02-20 2013-10-31 Laboratory Corporation Of America Holdings Diagnostic Methods and Markers for Bacterial Vaginosis
US20170183716A1 (en) * 2012-02-20 2017-06-29 Laboratory Corporation Of America Holdings Diagnostic Methods and Markers for Bacterial Vaginosis
CN102719529A (en) * 2012-05-04 2012-10-10 辽宁科骏生物有限公司 Trichomonas vaginalis and candida albicans two-channel fluorescence PCR detection method and kit thereof
CN107099618A (en) * 2017-05-03 2017-08-29 上海速创诊断产品有限公司 A kind of LAMP primer composition thing and its related application for being used to detect urogenital tract pathogenic microorganisms
CN109487013A (en) * 2019-01-09 2019-03-19 中生方政生物技术股份有限公司 Herpes simplex virus I-type and II type detection marker, primed probe are to, kit and detection method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
李自刚等: "《生物检测技术》", 中国轻工业出版社 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113151308A (en) * 2021-05-10 2021-07-23 广东省农业科学院动物卫生研究所 Nucleic acid molecule, PCR primer pair and kit for detecting avian trichomonas TBP gene
CN113136438A (en) * 2021-05-14 2021-07-20 广东省农业科学院动物卫生研究所 Nucleic acid molecule, PCR primer pair and kit for detecting avian trichomonas beta-tubulin gene
CN113136438B (en) * 2021-05-14 2023-08-08 广东省农业科学院动物卫生研究所 Nucleic acid molecule, PCR primer pair and kit for detecting trichomonas fowl beta-tubulin gene
CN116179731A (en) * 2023-03-31 2023-05-30 斯贝福（北京）生物技术有限公司 Primer probe set and kit for detecting trichomonas muriaticus and detection method

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