CN1873025A - Fluorescence quantitative PCR kit for detecting eye chlamydia and Urealpasma parvum synchronistically - Google Patents
Fluorescence quantitative PCR kit for detecting eye chlamydia and Urealpasma parvum synchronistically Download PDFInfo
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Abstract
This invention discloses a fluorescent PCR test kit for quantitatively detecting Chlamydia trachomatis and Ureaplasma parvum simultaneously, which is composed of DNA lysis solution, fluorescent quantitative PCR reaction solution, and positive standard templates PU-UP and PU-CT and negative standard samples. The fluorescent quantitative PCR reaction solution comprises a pair of specific promers and a fluorescent probe of Ureaplasma parvum, and a pair of specific primers and a fluorescent probe of Chlamydia trachomatis. The test kit has such advantages as high specificity, high sensitivity, high accuracy, simple operation and good repeatability. The test kit can be used for detecting Chlamydia trachomatis and Ureaplasma parvum simultaneously, and can replace the traditional culture method and the enzyme linked immunosorbent assay (ELISA) detection method.
Description
Technical field
The present invention relates to a kind of sexually transmitted disease (STD) pathogen gene detection technique, relate in particular to a kind of synchronous detection chlamydia trachomatis and tiny urea substance quantitative fluorescent PCR (polymerase chain reaction) test kit, be applicable to chlamydia trachomatis and the synchronous qualitative and quantitative detection of tiny urea substance.
Background technology
Chlamydia trachomatis (Chlamydia trachomatis, CT) and tiny urea substance (Ureaplasmaparvum, UP) be the main pathogens of nongonococcal urethritis (or cervicitis), be the main pathogenic microbes that causes spontaneous abortion, also increasing by prostatitis and infertility that these two kinds of pathogenic infections are concurrent.Ureaplasma urealyticum is divided into two biological groups and 14 serotypes.These two biological groups can be identified from phenotype and heredity.But more and more evidences shows that it can be divided into two kinds, be Ureaplasma parvum (the tiny urea substance of called after, former biological group 1 comprises serotype 1,3,6,14) and Ureaplasmaurealyticum (biological group 2 in the past comprises serotype 2,4,5,7～13, keep original title ureaplasma urealyticum).Surely grow or cause that the overwhelming majority of infection is the tiny urea substance of biological group 1-at human urogenital tract.
Various in recent years reports show that the gonococcal urethritis sickness rate descends, and nongonococcal urethritis constantly rises, and occupies the first place of sexually transmitted disease (STD).Nongonococcal urethritis can have symptom or asymptomatic, and the sustainable existence of subclinical infection for many years.No matter symptom or asymptomatic is arranged, its consequence is serious equally: except that urethritis, eye conjunctivitis, can cause other reproductive organ inflammation, as epididymitis, prostatitis, vasitis, cervicitis, vaginitis, salpingitis and pelvic inflammatory disease etc., cause infertility and ectopic pregnancy at last, also can infect the baby and cause conjunctivitis and pneumonia.Men homosexuality person can suffer from rectitis and pharyngitis.Normal and the gonorrhoea of nongonococcal urethritis infects simultaneously.The former occurs the gonorrhoea symptom earlier, and after the treatment of anti-gonorrhoea, gonococcus is killed by penicillin, and chlamydozoan, mycoplasma still exist.Infecting 1-3 week sequela.The very easy clinically gonorrhoea that is mistaken as is not cured or is recurred.This shows that nongonococcal urethritis has caused great injury for people's physical and mental health, must find correct effective diagnostic method.
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; Yin Ci Qi treats 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 synchronous detection chlamydia trachomatis and tiny urea substance is provided.
The present invention is by the following technical solutions:
One, PCR test kit
This test kit comprises: a) dna cleavage liquid, b) fluorescence quantitative PCR reaction solution, c) UP standard positive template pU-UP, d) CT standard positive template pU-CT, e) negative quality control standard product;
Fluorescence quantitative PCR reaction solution contain the chlamydia trachomatis specific primer to and fluorescent probe, and tiny urea substance Auele Specific Primer to and fluorescent probe;
Tiny urea substance (UP):
Forward primer is 5 '-CATTGATGTTGCACAAGGAGAAA-3 ',
Reverse primer is 5 ' TTAGCACCAACATAAGGAGCTAAATC-3 ';
The fluorescent probe sequence is 5 '-TTGACCACCCTTACGAG-3 ';
Fluorescent probe 5 ' end mark report fluorophor HEX, the non-luminous quenching group mark of 3 ' end mark, and connection small recesses binding substances-MGB group (TaqMan MGB probe), can improve the annealing temperature (Tm value) of probe greatly, standard positive template pU-UP contains the pUCm-T carrier that the nucleotide fragments of 111 base pairs of tiny urea substance high conservative gene-urase gene constitutes, and this carrier can be bred in bacillus coli DH 5 alpha.
Chlamydia trachomatis (CT)
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: 50mmo1/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 comprises: PCR 10 * buffer 3.0 μ l, each 1.0 μ l of 10 μ mol/L UP forward primers and reverse primer, each 0.7 μ l of 10 μ mol/L CT forward primers and reverse primer, UP 5 μ mol/L fluorescent probes 1.0 μ l, CT5 μ mol/L fluorescent probe 0.7 μ l, 25mmol/L MgCl
23 μ l, 10mmol/LdNTPs 0.7 μ l, 2.5U/ μ l HOTSTART Taq archaeal dna polymerase 0.6 μ l, aseptic double-distilled water 5.6 μ l, reaction solution cumulative volume 18 μ l.The nucleotide sequence of fluorescent probe shown in being wherein, UP fluorescent probe 5 ' end mark HEX, 3 ' end mark MGB, CT fluorescent probe 5 ' end flag F AM, 3 ' hold mark TAMRA.
C) UP standard positive template pU-UP
UP standard positive template pU-UP contains the pUCm-T recombinant plasmid that 1 nucleotide fragments of tiny urea substance high conservative gene ureE gene 11 constitutes, this recombinant plasmid transformed bacillus coli DH 5 alpha propagation back alkaline lysis method of extracting, through DNA purification kit purifying, quantitatively and be diluted to 10 with spectrophotometric instrumentation A260
9Copy/μ l ,-20 ℃ of preservations.Storing concentration is 10
9Copy/μ l carries out the serial dilution of 10 times of gradients before the use.
D) CT standard positive template pU-CT
CT standard positive template pU-CT contains the pUCm-T recombinant plasmid that 73 nucleotide fragments of chlamydia trachomatis trp gene constitute, this recombinant plasmid transformed bacillus coli DH 5 alpha propagation back alkaline lysis method of extracting, through DNA purification kit purifying, quantitatively and be diluted to 10 with spectrophotometric instrumentation A260
9Copy/μ l ,-20 ℃ of preservations.Storing concentration is 10
9Copy/μ l carries out the serial dilution of 10 times of gradients before the use.
E) negative quality control standard product
Negative quality control standard product are aseptic double-distilled water.
The principle of work of this test kit:
In chlamydia trachomatis of detection by quantitative simultaneously and rapidly provided by the invention and tiny urea substance PCR kit for fluorescence quantitative, the underlined 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, the 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 and tiny urea substance 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 synchronous detection chlamydia trachomatis provided by the invention and tiny urea substance PCR kit for fluorescence quantitative, at the singularity in chlamydia trachomatis and the detection of tiny urea substance, 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 chlamydia trachomatis and the synchronous detection by quantitative of tiny urea substance.Pass through prioritization scheme, experiment repeatedly, synchronous detection chlamydia trachomatis and tiny urea substance fluorescence quantifying PCR method have been set up, and develop synchronous detection chlamydia trachomatis and tiny urea substance 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 and tiny urea substance.
Two, using method of the present invention comprises the following steps:
A) be 10 to storing concentration
9Copy/μ l standard positive template pU-UP and pU-CT carry out 10 times 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 UP with dna cleavage liquid, CT DNA;
C) get b respectively) two kinds of 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 respective standard product relatively.
The present invention compared with prior art has the following advantages and effect:
1, specificity is good, and is highly sensitive, quantitatively accurately;
2, detection speed is fast, and only 1 hour, add the extraction preparation of sample DNA, only need 2 hours altogether;
3, use step simple, repeatable high;
But 4 synchronous detection chlamydia trachomatises and tiny urea substance;
5, can carry out high-throughout sample detection simultaneously.
This test kit can carry out synchronous detection by quantitative to chlamydia trachomatis and tiny urea substance, 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: test kit is formed and preparation
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 3.0 μ l, each 1.0 μ l of 10 μ mol/L UP forward primers and reverse primer, each 0.7 μ l of 10 μ mol/L CT forward primers and reverse primer, UP 5 μ mol/L fluorescent probes 1 μ l, CT5 μ mol/L fluorescent probe 0.7 μ l, 25mmol/L MgCl
23 μ l, 10mmol/LdNTPs 0.7 μ l, 2.5U/ μ l HOTSTART Taq archaeal dna polymerase 0.6 μ l, aseptic double-distilled water 5.6 μ l form.
D, standard positive template stock solution: concentration is 10
9Copy/μ l standard positive template pU-UP, concentration is 10
9Copy/μ l standard positive template pU-CT.
E, negative quality control standard product: be aseptic double-distilled water
Embodiment 2: use test kit synchronous detection chlamydia trachomatis and tiny urea substance
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 UPDNA and the b) each 1 μ l of UP positive criteria template of step dilution, get a) step gained CTDNA and the b) each 1 μ l of CT positive criteria template of step dilution, and establish negative control, add 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 synchronous detection wavelength is 556nm (UP-HEX) and 510nm (CT-FAM).
After the loop ends, the utilization instrument carries software, reads sample copy number to be checked.The result is: UP 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 12.11,15.62, and 18.33,22.98,26.59,30.02; CT 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 12.61,15.92, and 19.06,23.25,27.34,31.47; 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, and traditional method can only separately be carried out for the detection of these two kinds of pathogenic agent, often the cycle long, the cost height.Therefore, use 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, a kind of synchronous detection chlamydia trachomatis and tiny urea substance PCR kit for fluorescence quantitative is characterized in that:
Form by dna cleavage liquid, fluorescence quantitative PCR reaction solution, standard positive template pU-UP, standard positive template pU-CT, negative quality control standard product; Fluorescence quantitative PCR reaction solution contain the chlamydia trachomatis specific primer to and fluorescent probe, and tiny urea substance Auele Specific Primer to and fluorescent probe;
①UP
Forward primer is 5 '-CATTGATGTTGCACAAGGAGAAA-3 ',
Reverse primer is 5 ＇-TTAGCACCAACATAAGGAGCTAAATC-3 ';
The fluorescent probe sequence is 5 '-TTGACCACCCTTACGAG-3 ';
Fluorescent probe 5 ' end mark report fluorophor HEX, the non-luminous quenching group mark of 3 ' end mark, and connection small recesses binding substances-MGB group, can improve the annealing temperature of probe greatly, standard positive template pU-UP contains the pUCm-T carrier that the nucleotide fragments of 111 base pairs of tiny urea substance high conservative gene-urase gene constitutes, and this carrier is bred in bacillus coli DH 5 alpha;
②CT
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 is bred in bacillus coli DH 5 alpha;
Described PCR is the polymerase chain reaction, and described UP is tiny urea substance, and described CT is a chlamydia trachomatis.
2, test kit according to claim 1 is characterized in that:
Fluorescence quantitative PCR reaction solution is by PCR10 * buffer, UP forward primer and the reverse primer of 10 μ mol/L, CT forward primer and the reverse primer of 10 μ mol/L, 5 μ mol/L UP fluorescent probes, 5 μ mol/L CT fluorescent probes, 25mmol/L MgCl
2, 10mmol/L dNTPs and aseptic double-distilled water are formed.
3, test kit according to claim 1 is characterized in that:
The nucleotides sequence of the ureE gene that UP standard positive template pU-UP comprises is classified as
CATTGATGTTGCACAAGGAGAAAAAATTCCTCGTAAGGGTGGTCAAGGNATGATTAAATCAGAMTTATTTATTATTAATAAAGTTGATTTAGCTCCTTATGTTGGTGCTAA；
N:G or A; M:T or C;
The nucleotides sequence of the trp gene that CT standard positive template pU-CT comprises is classified as
TTCAGTTGGGCCAGATCATGCCGAAATGCATGAGTCAGGACGAGCCTTTTATACATTAGCCACCGATGAAGAG。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101608239B (en) * | 2009-03-26 | 2011-11-16 | 重庆大学 | Animal chlamydia multiple sleeve type PCR detection kit and detection method thereof |
| CN102277429A (en) * | 2011-07-22 | 2011-12-14 | 广东凯普生物科技股份有限公司 | Gonococcus, chlamydia trachomatis and ureaplasma urealyticum detection kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1086739C (en) * | 1996-07-05 | 2002-06-26 | 中国科学院动物研究所 | Single tube synchronous three-step PCR checkout method for chlamydia trachomatis and Neisseria gonorrhoeae |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101608239B (en) * | 2009-03-26 | 2011-11-16 | 重庆大学 | Animal chlamydia multiple sleeve type PCR detection kit and detection method thereof |
| CN102277429A (en) * | 2011-07-22 | 2011-12-14 | 广东凯普生物科技股份有限公司 | Gonococcus, chlamydia trachomatis and ureaplasma urealyticum detection kit |
| CN102277429B (en) * | 2011-07-22 | 2013-01-09 | 广东凯普生物科技股份有限公司 | Gonococcus, chlamydia trachomatis and ureaplasma urealyticum detection kit |
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