CN1873023A - Fluorescence quantitative kit PCR for quick testing chlamydia trachomatis - Google Patents
Fluorescence quantitative kit PCR for quick testing chlamydia trachomatis Download PDFInfo
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- CN1873023A CN1873023A CN 200610018766 CN200610018766A CN1873023A CN 1873023 A CN1873023 A CN 1873023A CN 200610018766 CN200610018766 CN 200610018766 CN 200610018766 A CN200610018766 A CN 200610018766A CN 1873023 A CN1873023 A CN 1873023A
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Abstract
This invention discloses a fluorescent PCR test kit for quantitatively detecting Chlamydia trachomatis, which is composed of DNA lysis solution, fluorescent quantitative PCR reaction solution, positive standard template and negative standard sample. The fluorescent quantitative PCR reaction solution comprises forward and reverse primers and fluorescent probes. The test kit has such advantages as high specificity, high sensitivity, high accuracy, simple operation and good repeatability.
Description
Technical field
The present invention relates to a kind of sexually transmitted disease (STD) pathogen gene detection technique, (Chlamydia trachomatis, quantitative fluorescent PCR CT) (polymerase chain reaction) test kit is applicable to the CT qualitative and quantitative detection to relate in particular to a kind of rapid detection chlamydia trachomatis.
Background technology
CT is a kind of pathogenic microorganism, neither bacterium, neither virus.It can cause urinary tract infection, is beyond the gonorrhoea bacterium, comparatively common sexually transmitted disease (STD).The typical case of this urethritis performance is that urethra is scratchy, odynuria and misnicturition.In the women, also vaginitis, cervicitis can be arranged simultaneously, even cause pelvic inflammatory disease.
The women infects this disease also can cause infertile, ectopic pregnancy.The pregnant woman is ill, and its newborn infant infects through birth canal also can bring out eye conjunctivitis, or pneumonia takes place.The sustainable long period of chlamydia trachomatis infection needs treatment in time and fully, otherwise delays easily and recur.
The quantitative fluorescent PCR that development in recent years is got up (Fluorescence Quantitative PCR, FQ-PCR) technology is highly sensitive with it, speed is fast, advantages such as high specificity are at gene expression dose analysis (Nellemann C, Vinggaard AM, Dalgaard M, et al.Quantification of Antiandrogen EffectDetermined by LightCycler Technology[J] .Toxicology, 2001,163:29-38), qualitative (the McGoldrick A of pathogenic agent, Lowings JP, Ibata G, et al.A Novel Approachto the Detection of Classical Swine Fever Virus by RT-PCR with aFluorogenic Probe (TaqMan) [J] .J.Virol.Methods, 1998,72:125-135.; Bhudevi B, Weinstock D.Fluorogenic RT-PCR assay (TaqMan) for Detectionand Classification of Bovine Viral Diarrhea Virus[J] .Vet.Microbiol., 2001,83:1-10.) and detection by quantitative (Desire N, Dehee A, Schneider V, et al.Quantification of Human Immunodeficiency Virus Type 1 Proviral Load bya TaqMan Real-Time PCR Assay[J] .J.Clin.Microbiol, 2001,39:1303-1310.; Martell M, Gomez J, Esteban JI, et al.High-ThroughputReal-Time Reverse Transcription-PCR Quantitation of Hepatitis C Virus RNA[J] .J.Clin.Microbiol, 1999,37:327-332.; Kearns AM, Turner AJL, Eltringham GJA, et al.Rapid Detection and Quantification of CMV DNA inUrine using Lightcycler-based Real-time PCR[J] .J.Clin.Virol, 2002,24:131-134; Florence KP, Glaucia PB, Mireille S, et al.Quantitationof HCV RNA using real-time PCR and fluorimetry[J] .J.Virol.Methods, 2001,95:111-119.) etc. the aspect be used widely, and become the quantitative main method of current viral nucleic acid, domestic existing at present test kit listing about third liver, hepatitis B, mycoplasma, acquired immune deficiency syndrome (AIDS), tuberculosis detection by quantitative.
Culture method and serological method traditional on the clinical detection have shortcomings such as sensitivity is low, poor specificity, false positive, time and effort consuming; Though conventional PCR method has easy, quick, sensitive advantage, have can not accurate quantification and the PCR aftertreatment produce the problems such as false positive that pollution causes; Therefore according to treating accurate, sensitive, quick, the free of contamination Clinical Laboratory method of exploitation.
Summary of the invention
The objective of the invention is to overcome the shortcoming and defect that prior art exists, the PCR kit for fluorescence quantitative of a kind of rapid detection chlamydia trachomatis is provided.
The present invention is by the following technical solutions:
One, PCR test kit
This test kit comprises that this test kit comprises: a) dna cleavage liquid, b) fluorescence quantitative PCR reaction solution, c) standard positive template, d) negative quality control standard product;
Fluorescence quantitative PCR reaction solution contains primer and fluorescent probe, and primer is divided into forward primer and reverse primer;
Forward primer is 5 '-TTCAGTTGGGCCAGATCATG-3 ',
Reverse primer is 5 '-CTCTTCATCGGTGGCTAATGTATAAA-3 ';
The fluorescent probe sequence is 5 '-AGGCTCGTCCTGACTCATGCATTTCG-3 ',
Fluorescent probe 5 ' end flag F AM, 3 ' end mark TAMRA group, standard positive template pU-CT is the pUCm-T carrier that contains the nucleotide fragments formation of 73 base pairs of chlamydia trachomatis high conservative gene trp gene, and this carrier can be bred in bacillus coli DH 5 alpha.
Specifically:
A) dna cleavage liquid
Dna cleavage liquid comprises: 50mmol/L NaOH, and 10mmol/L Tris-HCl, pH 8.0, and volume fraction is 1%Triton X-100, and volume fraction is 1%NP-40,0.5mmol/L EDTA pH 8.0.
B) fluorescence quantitative PCR reaction solution
Fluorescence quantitative PCR reaction solution is by forward primer and reverse primer, fluorescent probe, PCR 10 * buffer solution, MgCl
2Solution, dNTPs solution, Taq archaeal dna polymerase and aseptic double-distilled water are formed; In a concrete scheme of the present invention, the fluorescent quantitation reaction solution is by PCR 10 * buffer 2 μ l, 10 μ mol/L forward primers and reverse each 1.0 μ l of primer, 5 μ mol/L fluorescent probes, 0.8 μ l, 25mmol/L MgCl
22 μ l, 10mmol/L dNTPs 0.4 μ l, 2.5U/ μ l HOTSTART Taq archaeal dna polymerase 0.4 μ l, aseptic double-distilled water 10.4 μ l form, reaction solution cumulative volume 18 μ l.The nucleotide sequence of fluorescent probe shown in being wherein, fluorescent probe 5 ' end flag F AM, 3 ' end mark TAMRA.
C) standard positive template
Standard positive template pU-CT contains the pUCm-T carrier that 73 nucleotide fragments of chlamydia trachomatis trp gene constitute, and this carrier can be bred in bacillus coli DH 5 alpha.Storing concentration is 10
9Copy/μ l, serial dilution before using.Testing used standard substance is the plasmid pU-CT that contains the purpose amplified fragments, and alkaline lysis method of extracting is used in this plasmid transformation escherichia coli DH5 α propagation back, through DNA purification kit purifying, with spectrophotometric instrumentation A
260Quantitatively and be diluted to 10
9Copy/μ l ,-20 ℃ of preservations.
D) negative quality control standard product
Negative quality control standard product are aseptic double-distilled water.
The principle of work of this test kit:
Detect in the chlamydia trachomatis PCR kit for fluorescence quantitative at fast quantification provided by the invention, there are two ends to be marked with the specificity fluorescent probe of fluorophor, when probe is complete, two groups distance on space structure is close mutually, the fluorescence that 5 ' end reporter group produces is because FRET (fluorescence resonance energy transfer) (FRET) and by the group cancellation of going out of 3 ' end quenching, so there is not the variation of fluorescent signal in the system.In PCR annealing and extension process, probe combines with template specificity, extension along with primer, HOTSTART Taq archaeal dna polymerase utilizes its 5 ' → 3 ' circumscribed activity that probe cutting is discharged reporter group, destroyed the FRET (fluorescence resonance energy transfer) (FRET) between two groups like this, the photofluorometer that the fluorescence that reporter group discharged can be built in the detection by quantitative instrument detects, and the accumulation volume of the increase of fluorescence volume and PCR product is proportionlity.To chlamydia trachomatis quantitatively can comparing draws by the circulation thresholding (Ct, Threshold Cycle) with standard substance.The Ct value is in the PCR process, and the accumulation of fluorescence volume surpasses the circulation number of substrate fluorescence volume, and Ct value and initial modulus are the certain proportion relation, and the Ct value is more little, and the starting template number is many more, and on the contrary, the Ct value is big more, and the starting template number is few more.Utilize the Ct value of positive gradient standard form to make typical curve, can accurately measure the initial copy number of this sample again according to the Ct value of testing sample.
In detection chlamydia trachomatis PCR kit for fluorescence quantitative provided by the invention, at the singularity in the chlamydia trachomatis detection, different target fragments is carried out reaction system, optimization as primer and concentration and probe concentration, Mg2+ concentration, annealing temperature etc., and FQ-PCR technology (fluorescent quantitative PCR technique) and detection by quantitative system combined, use it for the chlamydia trachomatis detection by quantitative.Pass through prioritization scheme, detection chlamydia trachomatis fluorescence quantifying PCR method has been set up in experiment repeatedly, and develops and detect the chlamydia trachomatis PCR kit for fluorescence quantitative, the sensitivity of this test kit can detect 10 copies in each reaction system, can satisfy the requirement of quick diagnosis chlamydia trachomatis.
Two, using method of the present invention comprises the following steps:
A) be 10 to storing concentration
9Copy/μ l standard positive template pU-CT carries out serial dilution, preparation positive criteria product, and it is quantitative to standard substance to measure A260 with ultraviolet spectrophotometer;
B) from sample to be measured, extract DNA with dna cleavage liquid;
C) get b respectively) positive criteria product in a) step of the DNA in the step and the serial dilution of same amount join in the PCR reaction system that contains HOTSTART Taq archaeal dna polymerase and fluorescent quantitation reaction solution and carry out the PCR detection with the fluorescent quantitation detector;
D), the initial copy number of testing sample is carried out quantitatively by the circulation thresholding Ct value of testing sample and standard substance relatively.
The present invention compared with prior art has the following advantages and effect:
1, quantitatively accurately;
2, detection speed is fast, and only 1 hour, add the extraction preparation of sample DNA, only need 2 hours altogether;
3, specificity is good, and is highly sensitive;
4, step is simple, and is repeatable high;
5, can carry out high-throughout sample detection simultaneously.
The fast quantification that provides in the present invention detects the chlamydia trachomatis PCR kit for fluorescence quantitative can carry out detection by quantitative to chlamydia trachomatis, and the alternative traditional culture method always continued to use and ELISA (enzyme linked immunosorbent assay) diagnostic method.
Embodiment
Below in conjunction with concrete embodiment, further set forth the present invention.Be to be understood that, these embodiments only are used to the present invention is described and are not used in restriction the scope of protection of present invention, unreceipted concrete experiment condition and method in the following example, usually according to normal condition as chief editors such as J. Sa nurse Brookers, Science Press, 1992, molecular cloning experiment guide (second edition); D.L. Spector etc., Science Press, 2001, cell experiment guide; Lv Hongsheng, Science Press, 1982, or connect the condition of advising according to manufacturer.
Embodiment 1: the chlamydia trachomatis PCR kit for fluorescence quantitative is formed and preparation
Reagent is formed:
A, DNA extraction reagent (lysate)
Be oneself preparation, lysate: 50mmol/L NaOH, 10mmol/L Tris-HCl, pH 8.0, and volume fraction is 1%Triton X-100, and volume fraction is 1% NP-40,0.5mmol/L EDTA pH 8.0.
B, fluorescent PCR 10 * Buffer form:
500mM?KCl、100mM?Tris-HCl(PH9.0?25℃)、
1.0％Triton?X-100；
C, fluorescence quantitative PCR reaction solution: PCR 10 * buffer 2 μ l, each 1.0 μ l (10 μ mol/L) of forward primer and reverse primer, fluorescent probe 0.8 μ l (5 μ mol/L), MgCl
22 μ l (25mmol/L), dNTPs 0.4 μ l (10mmol/L), HOTSTART Taq archaeal dna polymerase 0.4 μ l (2.5U/ μ l), aseptic double-distilled water 10.4 μ l.The fluorescent probe working concentration is 5 μ mol/L.
D, standard positive template stock solution: concentration is 10
9Copy/μ l standard positive template pU-CT.
E, negative quality control standard product: be aseptic double-distilled water
Embodiment 2: the PCR kit for fluorescence quantitative that detects with the chlamydia trachomatis fast quantification detects chlamydia trachomatis
A, add the 1ml stroke-physiological saline solution in the sample test tube, fully concussion shakes up, go in the 1.5ml centrifuge tube, and the centrifugal 5min of 10000g, repeated washing is 1 time again.Precipitation directly adds the abundant mixing of 50 μ l DNA extraction liquid, boiling water bath 10min, and the centrifugal 5min of 10000g gets supernatant liquor 2 μ l and does the PCR reaction.
B, be 10 with positive criteria template (reagent d) serial dilution
8Copy/μ l, 10
7Copy/μ l, 10
6Copy/μ l, 10
5Copy/μ l, 10
4Copy/μ l, 10
3Copy/μ l.
C, get each 18 μ l of fluorescence quantitative PCR reaction solution (reagent c) respectively, get a) step gained DNA and the b) each 2 μ l of positive criteria template of step dilution, and establish negative control, and adding different PCR reaction tubess respectively, the parallel PCR that is detects on the fluorescent quantitation detector.Cycling condition is: 95 ℃ of pre-sex change 400s; 95 ℃ of 10s, 58 ℃ of 40s, 40 circulations of increasing.Carry out when the program of fluoroscopic examination is arranged on each round-robin second EOS, the detection wavelength is 510nm.
After the loop ends, the utilization instrument carries software, reads sample copy number to be checked.The result is: standard positive template 10
8Copy/μ l, 10
7Copy/μ l, 10
6Copy/μ l, 10
5Copy/μ l, 10
4Copy/μ l, 10
3The Ct value of copy/μ l is respectively 12.81,16.24,20.12,23.27,25.17,27.89, and negative control is 0;
Revision test is 3 times repeatedly, obtains the Ct value and carries out statistical analysis P＞0.05, and the data difference nonsignificance illustrates that the detected result between its different batches has comparability, has good repeatability.
Can illustrate from above-mentioned example, the fluorescence quantifying PCR method quantitative repeatability is good, quantitatively accurately, and, fluorescent quantificationally PCR detecting kit only needed just can finish in 2 hours to the detection of sample, and traditional cell culture method needs just can finish about 1 week approximately, therefore, uses this test kit to shorten detection time greatly.
The operation of this test kit only needs 1 people can finish whole quantitative work process, once can detect 32-384 (by the model decision of detection by quantitative instrument) sample, has so also reduced waste of manpower resource.
Claims (3)
1, the PCR kit for fluorescence quantitative of a kind of rapid detection chlamydia trachomatis is characterized in that:
Form by dna cleavage liquid, fluorescence quantitative PCR reaction solution, standard positive template, negative quality control standard product;
Fluorescence quantitative PCR reaction solution contains primer and fluorescent probe, and primer is divided into forward primer and reverse primer;
Forward primer is 5 '-TTCAGTTGGGCCAGATCATG-3 ',
Reverse primer is 5 '-CTCTTCATCGGTGGCTAATGTATAAA-3 ';
The fluorescent probe sequence is 5 '-AGGCTCGTCCTGACTCATGCATTTCG-3 ',
Fluorescent probe 5 ' end flag F AM group, 3 ' end mark TAMRA group, standard positive template pU-CT is the pUCm-T carrier that contains the nucleotide fragments formation of 73 base pairs of chlamydia trachomatis high conservative gene trp gene, and this carrier can be bred in bacillus coli DH 5 alpha;
Described PCR is the polymerase chain reaction, and described CT is a chlamydia trachomatis.
2, PCR test kit according to claim 1 is characterized in that:
Fluorescence quantitative PCR reaction solution is by PCR10 * buffer, the forward primer of 10 μ mol/L and reverse primer, 5 μ mol/L fluorescent probes, 25mmol/L MgCl
2, 10mmol/L dNTPs and aseptic double-distilled water are formed.
3, PCR test kit according to claim 1.The nucleotides sequence that it is characterized in that the trp gene that standard positive template pU-CT comprises is classified as:
TTCAGTTGGGCCAGATCATGCCGAAATGCATGAGTCAGGACGAGCCTTTTATACATTAGCCACCGATGAAGAG。
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101285037B (en) * | 2007-04-12 | 2010-05-19 | 中国科学院武汉病毒研究所 | Process for efficiently cracking chlamydia cell wall |
| CN101586162B (en) * | 2009-06-10 | 2011-02-09 | 戴立忠 | Method of extracting target nucleic acid and performing PCR amplification |
| CN102094073A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆临床检验中心有限公司 | Fluorescent polymerase chain reaction (PCR) kit for detecting chlamydia trachomatis infection by SYBR Green method |
| CN101497926B (en) * | 2008-12-23 | 2012-02-29 | 武汉大学 | Reagent kit for rapidly and synchronously detecting Chlamydi trachomatis, Urealpasma parvum and Ureaplasma urealyticum |
| CN103060452A (en) * | 2013-01-10 | 2013-04-24 | 湖南圣湘生物科技有限公司 | Kit for detecting chlamydia trachomatis (CT) |
| CN103993085A (en) * | 2014-05-25 | 2014-08-20 | 浙江省医疗器械研究所 | Specific primers, probe and method used for detecting chlamydia trachomatis (CT) |
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| CN111118172A (en) * | 2020-01-06 | 2020-05-08 | 中国农业科学院植物保护研究所 | Specific probe of aleyrodids Asiai II3 cryptophyte transient receptor ion channel gene and application thereof |
| CN112779248A (en) * | 2021-02-04 | 2021-05-11 | 杭州遂曾生物技术有限公司 | Chlamydia trachomatis integrated nucleic acid detection card box |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6096501A (en) * | 1997-11-04 | 2000-08-01 | Becton Dickinson And Company | Assay for Chlamydia trachomatis by amplification and detection of Chlamydia trachomatis crytpic plasmid |
| JP2004261017A (en) * | 2003-02-27 | 2004-09-24 | Arkray Inc | Method for detecting chlamydia trachomatis and kit for the same |
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2006
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101285037B (en) * | 2007-04-12 | 2010-05-19 | 中国科学院武汉病毒研究所 | Process for efficiently cracking chlamydia cell wall |
| CN101497926B (en) * | 2008-12-23 | 2012-02-29 | 武汉大学 | Reagent kit for rapidly and synchronously detecting Chlamydi trachomatis, Urealpasma parvum and Ureaplasma urealyticum |
| CN101586162B (en) * | 2009-06-10 | 2011-02-09 | 戴立忠 | Method of extracting target nucleic acid and performing PCR amplification |
| CN102094073A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆临床检验中心有限公司 | Fluorescent polymerase chain reaction (PCR) kit for detecting chlamydia trachomatis infection by SYBR Green method |
| CN103060452A (en) * | 2013-01-10 | 2013-04-24 | 湖南圣湘生物科技有限公司 | Kit for detecting chlamydia trachomatis (CT) |
| CN103060452B (en) * | 2013-01-10 | 2014-03-05 | 湖南圣湘生物科技有限公司 | Kit for detecting chlamydia trachomatis (CT) |
| CN103993085A (en) * | 2014-05-25 | 2014-08-20 | 浙江省医疗器械研究所 | Specific primers, probe and method used for detecting chlamydia trachomatis (CT) |
| CN104561378A (en) * | 2014-12-24 | 2015-04-29 | 华美生物工程有限公司 | Real-time fluorescent multiple PCR (Polymerase Chain Reaction) rapid detection kit for common pathogens leading to ocular infection |
| CN104561378B (en) * | 2014-12-24 | 2016-09-07 | 华美生物工程有限公司 | The real-time fluorescence multiple PCR fast detection kit of ocular infection common causative |
| CN110157820A (en) * | 2019-04-28 | 2019-08-23 | 深圳市国赛生物技术有限公司 | A kind of primer, probe and kit detecting chlamydia trachomatis |
| CN111118172A (en) * | 2020-01-06 | 2020-05-08 | 中国农业科学院植物保护研究所 | Specific probe of aleyrodids Asiai II3 cryptophyte transient receptor ion channel gene and application thereof |
| CN112779248A (en) * | 2021-02-04 | 2021-05-11 | 杭州遂曾生物技术有限公司 | Chlamydia trachomatis integrated nucleic acid detection card box |
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