CN113337624A - Composition and method for simultaneously detecting chlamydia trachomatis, neisseria gonorrhoeae and mycoplasma urealyticum - Google Patents
Composition and method for simultaneously detecting chlamydia trachomatis, neisseria gonorrhoeae and mycoplasma urealyticum Download PDFInfo
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- 241000606153 Chlamydia trachomatis Species 0.000 title claims abstract description 32
- 241000588652 Neisseria gonorrhoeae Species 0.000 title claims abstract description 32
- 229940038705 chlamydia trachomatis Drugs 0.000 title claims abstract description 32
- 239000000203 mixture Substances 0.000 title claims abstract description 18
- 241000204031 Mycoplasma Species 0.000 title claims abstract description 13
- 238000000034 method Methods 0.000 title claims abstract description 11
- 244000052769 pathogen Species 0.000 claims abstract description 17
- 230000001717 pathogenic effect Effects 0.000 claims abstract description 9
- 238000006243 chemical reaction Methods 0.000 claims abstract description 8
- 238000003753 real-time PCR Methods 0.000 claims abstract description 7
- 239000000523 sample Substances 0.000 claims description 38
- WCKQPPQRFNHPRJ-UHFFFAOYSA-N 4-[[4-(dimethylamino)phenyl]diazenyl]benzoic acid Chemical compound C1=CC(N(C)C)=CC=C1N=NC1=CC=C(C(O)=O)C=C1 WCKQPPQRFNHPRJ-UHFFFAOYSA-N 0.000 claims description 9
- 238000005070 sampling Methods 0.000 claims description 4
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 claims description 4
- 241000202921 Ureaplasma urealyticum Species 0.000 claims description 3
- 239000003086 colorant Substances 0.000 claims description 2
- 230000003321 amplification Effects 0.000 abstract description 6
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 6
- 239000007850 fluorescent dye Substances 0.000 abstract description 5
- 239000000975 dye Substances 0.000 abstract description 3
- 230000002708 enhancing effect Effects 0.000 abstract description 2
- 238000003752 polymerase chain reaction Methods 0.000 abstract 5
- 108091093037 Peptide nucleic acid Proteins 0.000 abstract 3
- 208000019802 Sexually transmitted disease Diseases 0.000 description 8
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- 210000003905 vulva Anatomy 0.000 description 1
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Abstract
The invention discloses a composition and a method for simultaneously detecting chlamydia trachomatis, neisseria gonorrhoeae and mycoplasma urealyticum, wherein primers of three screened pathogens and three PNA (peptide nucleic acid) fluorescent probes marked with different dyes and pathogen specific primers are used for simultaneously carrying out multiple real-time PCR (polymerase chain reaction) in the same reaction tube. According to the invention, by using three PNA fluorescent probes marked with different dyes and pathogen specific primers, CT, UU and NG can be simultaneously detected in real time in a single PCR tube. The Tm's of the PCR primers are similar, with no significant overlap of the 3' ends, enhancing multiplex amplification. During the PCR reaction, different fluorescent signals are detected and distinguished through different fluorescent channels of a PCR instrument, so that different pathogens are identified.
Description
Technical Field
The invention belongs to the field of nucleic acid detection, and particularly relates to a composition and a method for simultaneously detecting chlamydia trachomatis, neisseria gonorrhoeae and mycoplasma urealyticum.
Background
Sexually transmitted diseases are a group of diseases that have sexual contact as the primary mode of transmission. In general, there are three main routes of transmission for sexually transmitted diseases: direct contact transmission, indirect contact infection and placental tract infection. Due to the different pathogenic characteristics of venereal disease, some infected patients are often asymptomatic or asymptomatic and are therefore easily overlooked, and many patients do not go to the hospital until complications arise (Tsevat DG, 2017). Therefore, the rate of missed diagnosis is very high, especially in women. Establishing a rapid, accurate and reliable laboratory diagnosis is very important for early intervention and treatment of sexually transmitted diseases. The incidence of Sexually Transmitted Disease (STD) is still high in most countries, and venereal disease has also rapidly prevailed in china during the last two decades. Of these, gonococcal and non-gonococcal urethritis remain among the most common sexually transmitted diseases. Of these, Chlamydia Trachomatis (CT) and Mycoplasma urealyticum (UU) are considered pathogens of non-gonococcal urethritis, while Neisseria Gonorrhoeae (NG) is representative of gonococci.
Early diagnosis of venereal disease is critical to early intervention and treatment of patients (Hollier l.m., 2003). It may not only provide further diagnostic basis, but may sometimes be the only diagnostic basis, especially in cases where the infected person has no clinical manifestations. Currently, diagnostic techniques for CT, NG and UU have been developed from direct microscopic detection (Nathan B, 2015), isolation culture, biochemical identification and antigen-antibody detection of pathogen nucleic acid diagnosis (Nye MB, 2009). Molecular biology techniques for early screening and diagnosis of sexually transmitted diseases mainly include enzyme linked immunosorbent assay (ELISA) (Oranje a., 1983), strand displacement amplification, transcription-mediated amplification, PCR, molecular beacons, etc. (Muzny CA, 2014). There are also a variety of real-time PCR kits available for detecting STD pathogens, however, most of the common fluorescent PCR kits can detect only one pathogen and have low detection efficiency.
Disclosure of Invention
1. The invention aims to provide a novel method.
The purpose of the present invention is to provide a composition which can simultaneously detect Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma urealyticum with high efficiency.
2. The technical scheme adopted by the invention is disclosed.
The invention discloses a composition for simultaneously detecting chlamydia trachomatis, neisseria gonorrhoeae and mycoplasma urealyticum, which comprises a primer pair and a probe;
the primer pair comprises:
the primer pair comprises:
chlamydia trachomatis primer set C1f: GCAAGATATCGAGTATGCGTTGTTAGG
C1r:TTCATTGTACTCATTAAACGAGCGG；
Neisseria gonorrhoeae primer set G1f: TATCGGAACGTACCGGGTAGC
G1r:GTATTACCGCGGCTGCTGGCA；
Mycoplasma urealyticum primer set U6f1: CCAAGTTGAAGTTACTTTCCCAGATG
U6r1:5-AGGACGGTCACCAGTATTTTTAATG；
The probe includes:
chlamydia trachomatis C1 probe Lys (DABCYL) -Lys-GCTAAACAGGTATAGA-Glu-first fluorophore;
n1 probe from neisseria gonorrhoeae:
lys (dabcyl) -Lys-GTCCATGGCAGTAGCC-Glu-second fluorophore;
ureaplasma urealyticum U2 probe: lys (DABCYL) -Lys-TCAATTAACGAGGACC-Glu-third fluorescent group, wherein the first fluorescent group, the second fluorescent group and the third fluorescent group emit light with different colors.
Preferably, the first, second and third fluorophores are selected from VIC, NED, ROX, texas Red or FAM.
Preferably, the probe is:
chlamydia trachomatis C1 probe:
Lys(DABCYL)-Lys-GCTAAACAGGTATAGA-Glu-VIC；
n1 probe from neisseria gonorrhoeae:
lys (DABCYL) -Lys-GTCCATGGCAGTAGCC-Glu-NED or Texas Red;
ureaplasma urealyticum U2 probe:
Lys(DABCYL)-Lys-TCAATTAACGAGGACC-Glu-FAM。
the invention also discloses a kit for simultaneously detecting the chlamydia trachomatis, the neisseria gonorrhoeae and the mycoplasma urealyticum, which comprises the composition.
The invention also discloses a method for simultaneously detecting the chlamydia trachomatis, the neisseria gonorrhoeae and the mycoplasma urealyticum, which comprises the following steps:
(1) sampling, and extracting the genomic DNA of the sample;
(2) and (3) carrying out fluorescent quantitative PCR reaction by using the composition by using a sample as a template to obtain a quantitative curve, and observing whether the quantitative curve has fluorescent signals corresponding to the C1 probe of the chlamydia trachomatis, the N1 probe of the neisseria gonorrhoeae and the U2 probe of the mycoplasma urealyticum, wherein if yes, the corresponding pathogen is positive.
3. The technical effect produced by the invention.
The invention can simultaneously detect CT, UU and NG in real time in a single PCR tube by using three fluorescent probes marked with different dyes and pathogen specific primers. The Tm's of the PCR primers are similar, with no significant overlap of the 3' ends, enhancing multiplex amplification. During the PCR reaction, different fluorescent signals are detected and distinguished through different fluorescent channels of a PCR instrument, so that different pathogens are identified. The whole process of PCR amplification and product analysis is carried out under the condition of a single tube, and the whole process of the experiment only needs to open the reaction tube once when a sample is added, so that the external pollution can be prevented. In each cycle of PCR, real-time dynamic detection of the amplified product and automatic analysis of the result can be achieved. The multiplex real-time PCR has high specificity and high sensitivity, the detection limit is about 10 copies, and the detection standard of the kit is met.
Drawings
FIG. 1 is a quantitative curve of multiplex real-time PCR in example 1.
FIG. 2 is a photograph of gel electrophoresis in example 1.
FIG. 3 is a photograph of gel electrophoresis in example 2.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The present invention is further illustrated by the following specific examples, which are not intended to be limiting. The experimental methods and reagents of the formulations not specified in the examples are all prepared according to conventional conditions such as Sambrook et al, molecular cloning: conditions described in the test Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or conditions suggested by the manufacturer.
Except for special instructions, the PCR reagent and the materials used in the experiment are all reagents originally used for medical treatment by manufacturers. The fluorescent PCR instruments were ABI7000, ABI7300 and ABI 7500. Primers were all purchased from Tech Dragon Hong Kong. All primers were desalted and grade purified. The fluorescent probe was a PNA probe ordered from PANAGENE. All PCR amplification products were confirmed by sequencing to confirm the analysis.
Example 1
1. Design and Synthesis of primers and probes
The CT/NG/UU genomic sequence was found from GeneBank and PNA probes and PCR primers for the nucleic acid amplification region were searched and designed using Primer Express 2.0 software. The design mainly considers the Tm value and the secondary structure of the primer, the TM value is about 60 ℃, and the target gene is located in a conserved region. The sequences of the finally designed primers and probes are shown in Table 1.
Primers and probes designed in Table 1
TABLE 2 fluorescent dye combinations useful for triple PCR
ROX can be replaced with Texas Red.
2. Sampling, extraction of genomic DNA
Multiplex real-time PCR assays are aimed at detecting the presence of CT, NG or UU in cervical, vaginal and male urethral swab samples as well as urine samples, the number of each sample: n is 200. The different sampling methods are as follows: male: in acute stage of male uremia, purulent secretion is obtained by conventional sterilized cotton, and in non-acute stage, a cotton swab is gently inserted into urethra
The male may take up 1-2 hours before urine secretion. The prostate massage liquid can be used for people with prostate symptoms. A semen sample is taken. Female: an adult female patient first swabs cervical secretions with a sterile strip, then inserts another strip or sterile cotton swab into the cervix for about a few seconds, and then gently swirls and removes the secretions. Purulent secretions from the vulva are needed for infants and women.
100 μ L of sample was added to appropriate tubes with caps, centrifuged at maximum speed (>10000rpm) for 5-10 minutes, and using a micropipette with an inserted aerosol barrier tip, the supernatant was carefully removed from each tube and discarded without disturbing the pellet, and the tips were changed between tubes. Thereafter, 500. mu.L of washing solution (PBS buffer) was added to each tube. Vortexed vigorously and centrifuged at 10000rpm for 30s, and the supernatant removed from each tube and discarded, if necessary, the procedure can be repeated. The pellet was resuspended in 50. mu.L of DNA eluate and incubated at 100 ℃ for 10 minutes. The tube was centrifuged at 12000rpm for 1 minute, and the supernatant, which contained the DNA to be amplified, was taken. If amplification is not performed on the same day as the extraction, the treated sample may be stored at 2-8 ℃ for up to 5 days, or frozen at-20 °/-80 ℃.
3. Multiplex real-time PCR
And (3) PCR system: primer 0.4. mu.M, FAM probe 100nM, NED/VIC probe 200nM, Mg2+4.0mM, dNTP 0.2mM, buffer 1.0 Xand Taq enzyme 0.5. mu.L. Sample DNA. A fluorescent PCR instrument: ABI 7500. PCR cycling conditions: 2min at 50 ℃, 2min at 94 ℃, 20s at 95 ℃ and 60s at 60 ℃; 40 cycles. As shown in FIG. 1, the DNA sample used in FIG. 1 was a clinical sample CT/NG/UU mixture.
4. The results of gel electrophoresis are shown in FIG. 2. The DNA samples used in FIG. 2 (from left to right) are clinical samples CT/NG/UU mixture (2&10), NG/UU mixture (3&11), CT/UU mixture (4&12), CT/NG mixture (5&13) and UU (6&14), NG (7&15) and CT (8&16) single copies, lane M is marker, and lanes 1 and 9 are NTC (Non-Template Control blank).
Example 2
Plasmid DNA of three pathogens, namely NG, CT and UU, is prepared and diluted by a concentration gradient from 1 copy to 10000 copies. The mixed primers of three pathogens are used for amplifying the DNA of a single pathogen, and the PCR reaction conditions and the gel electrophoresis system are consistent with those of the example 1.
FIG. 3(A and B) shows the sensitive detection electrophoresis of the DNA of three pathogens, NG, CT and UU, respectively. The DNA copy number gradients for all three pathogens were 1 copy, 10 copies and 100 copies, respectively (NG1, NG10, NG100 represent 1, 10, 100 copies of template DNA in the reaction, respectively). The result shows that the detection sensitivity of the multiplex PCR to NG is 10 copies, CT is 1-10 copies, UU is 1-10 copies.
Claims (4)
1. A composition for simultaneously detecting Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma urealyticum comprises a primer pair and a probe;
the primer pair comprises:
chlamydia trachomatis primer set C1f: GCAAGATATCGAGTATGCGTTGTTAGG
C1r:TTCATTGTACTCATTAAACGAGCGG；
Neisseria gonorrhoeae primer set G1f: TATCGGAACGTACCGGGTAGC
G1r:GTATTACCGCGGCTGCTGGCA；
Mycoplasma urealyticum primer set U6f1: CCAAGTTGAAGTTACTTTCCCAGATG
U6r1:5-AGGACGGTCACCAGTATTTTTAATG；
The probe includes:
chlamydia trachomatis C1 probe Lys (DABCYL) -Lys-GCTAAACAGGTATAGA-Glu-first fluorophore;
n1 probe from neisseria gonorrhoeae:
lys (dabcyl) -Lys-GTCCATGGCAGTAGCC-Glu-second fluorophore;
ureaplasma urealyticum U2 probe: lys (DABCYL) -Lys-TCAATTAACGAGGACC-Glu-third fluorescent group, wherein the first fluorescent group, the second fluorescent group and the third fluorescent group emit light with different colors.
2. The composition of claim 1, wherein the first fluorophore, the second fluorophore, and the third fluorophore are selected from VIC, NED, ROX, texas Red, and FAM.
3. A kit for simultaneously detecting Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma urealyticum, characterized in that: comprising the composition of claim 1 or 2.
4. A method for simultaneously detecting chlamydia trachomatis, neisseria gonorrhoeae and mycoplasma urealyticum comprising the steps of:
(1) sampling, and extracting the genomic DNA of the sample;
(2) performing a fluorescence quantitative PCR reaction by using the composition of claim 1 or 2 as a template to obtain a quantitative curve, and observing whether the quantitative curve has fluorescence signals corresponding to the C1 probe of Chlamydia trachomatis, the N1 probe of Neisseria gonorrhoeae and the U2 probe of Mycoplasma urealyticum, if so, the corresponding pathogen is positive.
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Citations (4)
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|---|---|---|---|---|
| CN101041864A (en) * | 2007-03-06 | 2007-09-26 | 扬子江药业集团北京海燕药业有限公司 | Chlamydi trachomatis and Neisseria gonorrhoeae dual real-time fluorescence PCR detection method |
| CN101171260A (en) * | 2005-05-06 | 2008-04-30 | 阿普里拉股份有限公司 | PNA probe for detecting mycoplasma |
| CN101580535A (en) * | 2008-05-16 | 2009-11-18 | 太景生物科技股份有限公司 | Hcv protease inhibitors |
| CN109825615A (en) * | 2019-01-09 | 2019-05-31 | 中生方政生物技术股份有限公司 | Primer, probe, kit and the detection method of chlamydia trachomatis, gonococcus and urea substance Multiple detection |
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| CN101171260A (en) * | 2005-05-06 | 2008-04-30 | 阿普里拉股份有限公司 | PNA probe for detecting mycoplasma |
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| CN101580535A (en) * | 2008-05-16 | 2009-11-18 | 太景生物科技股份有限公司 | Hcv protease inhibitors |
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| Title |
|---|
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