CN110894534A - Primer, probe, kit and detection method for detecting mycoplasma genitalium - Google Patents
Primer, probe, kit and detection method for detecting mycoplasma genitalium Download PDFInfo
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- CN110894534A CN110894534A CN201911337119.7A CN201911337119A CN110894534A CN 110894534 A CN110894534 A CN 110894534A CN 201911337119 A CN201911337119 A CN 201911337119A CN 110894534 A CN110894534 A CN 110894534A
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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
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- 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
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Abstract
The invention relates to the technical field of medical detection, in particular to a primer and a probe for detecting mycoplasma genitalium, a kit and a detection method, wherein the primer and the probe comprise a primer pair with a nucleotide sequence shown in SEQ ID NO 1-2 and a probe with a nucleotide sequence shown in SEQ ID NO 3.
Description
Technical Field
The invention relates to the technical field of medical detection, in particular to a primer, a probe, a kit and a detection method for detecting mycoplasma genitalium.
Background
Mycoplasma Genitalium (MG) belongs to the Mycoplasma family, the class mollicutes, and is one of the pathogens responsible for non-gonococcal urethritis (NGU). In addition, MG is closely related to sexually transmitted diseases such as Pelvic Inflammatory Disease (PID), obstetric complications, prostatitis, epididymitis, and balanitis preputialis. In recent years, studies have found that MG infection may also be associated with the development of tumors and Human Immunodeficiency Virus (HIV).
2016 European guidelines for Mycoplasma genitalium infection indicate that the prevalence of Mycoplasma genitalium in male non-chlamydial non-gonococcal urethritis (NCNGU) is between 10% and 35%. In China, the infection rate of the mycoplasma genitalium in the outpatient service is 4.0-19.4%. In female mycoplasma genitalium infection, 30.4% are co-infected with gonorrhea, and 25.0% are co-infected with chlamydia. In addition, 2-33% of HIV infections are co-infected with Mycoplasma genitalium. In recent years, due to the prevalence of MG infections and the continuing advances in detection technology, MG has become one of the important pathogens in sexually transmitted diseases.
The MG detection method includes: isolation culture method, serological method and molecular biological method. Isolation culture is the "gold standard" for diagnosis of mycoplasma genitalium infection, but MG culture success rate is extremely low and the cycle is long. The lack of specificity of serological detection methods due to the cross-reactivity of MG with antigens of Mycoplasma pneumoniae (Mp) is limited in practical application. In recent years, the nucleic acid detection technology has been widely used in disease detection due to its advantages of high sensitivity, strong specificity, wide detection range, easy automation, and the like. At present, the domestic kit for detecting MG based on the PCR technology is less, the automation degree is not high, the detection time is longer, and the false negative is higher, so that the development of the kit which is high in sensitivity, short in detection time, low in false positive, simple, convenient and quick is a problem to be solved urgently.
Disclosure of Invention
In view of the above, the present invention aims to provide a primer and a probe for detecting mycoplasma genitalium, a kit and a detection method, wherein the kit has the advantages of high sensitivity, good specificity and short detection time for detecting mycoplasma genitalium.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a primer and a probe for detecting mycoplasma genitalium, which comprise the following components:
a primer pair of a nucleotide sequence shown in SEQ ID NO. 1-2 and a probe of a nucleotide sequence shown in SEQ ID NO. 3.
The invention includes all Mycoplasma genitalium genome sequences by comparing GeenBank databases, and designs a primer of a nucleotide sequence shown in SEQ ID NO 1-2 and a probe of a nucleotide sequence shown in SEQ ID NO 3 aiming at Mycoplasma genitalium outer membrane protein B genes, wherein:
SEQ ID NO:1：5’-AGCCTTACCACACCTTCGTAG-3’
SEQ ID NO:2：5’-TGGCATTCTTTGTTTGCATTGGAG-3’；
SEQ ID NO:3：5’-AGGGATCACCCATGCAGGGAGT-3’。
in some embodiments, the probe having the nucleotide sequence shown in SEQ ID NO. 3 is labeled with a fluorescent reporter group FAM at the 5 'end and a fluorescent quencher group BHQ1 at the 3' end.
The invention also provides a kit for detecting mycoplasma genitalium, which comprises the primer and the probe.
The kit provided by the invention also comprises a primer for detecting the nucleotide sequence shown in SEQ ID NO. 4-5 of the internal reference gene and a probe of the nucleotide sequence shown in SEQ ID NO. 6.
Primers of nucleotide sequences shown in SEQ ID NO. 4-5 and probes of nucleotide sequences shown in SEQ ID NO. 6 are designed aiming at human β globin gene, wherein:
SEQ ID NO:4：5’-TGGAACATATTTGACGGCTTT-3’
SEQ ID NO:5：5’-GATCTTAGCTACTACTGCCAA-3’
SEQ ID NO:6：5’-AAACCCACTGCATCACTCT-3’。
the 5 'end of the probe of the nucleotide sequence shown in SEQ ID NO. 6 is marked with a fluorescence reporter group ROX, and the 3' end is marked with a fluorescence quenching group BHQ 2.
The kit provided by the invention also comprises a reaction solution 1 and a reaction solution 2; the reaction solution 1 comprises a basic buffer solution, cations, an enhancer and enzymes; the reaction solution 2 comprises P300 and MnCl2。
In some embodiments, the base buffer is a Tris-HCl buffer containing BSA and the cation is Mn2+、K+Including one or more of glycerol, DMSO, Tween20, Tricine X-100, potassium acetate, and betaine, the enzyme being a bifunctional enzyme and/or a UNG enzyme.
The bifunctional enzyme is an enzyme with the functions of both RNA reverse transcription and DNA polymerase, and mainly comprises Tth DNA polymerase, TflDNA polymerase and related recombinant and improved enzymes.
The kit also comprises a negative quality control product and a positive quality control product, wherein the negative quality control product is a plasmid containing a human β globin gene segment, and the positive quality control product comprises a plasmid containing an MG outer membrane protein B gene segment and a plasmid containing a human β globin gene segment.
The invention also provides a detection method of the mycoplasma genitalium, which comprises the following steps: the kit of the invention is used for carrying out real-time fluorescent quantitative PCR detection on the DNA of a sample to be detected, and the S-shaped amplification curve is positive.
In some embodiments, the system for real-time fluorescent quantitative PCR comprises:
in some embodiments, the real-time fluorescent quantitative PCR system has a primer concentration of 20 to 35 pmol/. mu.l and a probe concentration of 10 to 20 pmol/. mu.l.
In some embodiments, the real-time fluorescent quantitative PCR procedure comprises:
2 minutes at 50 ℃;
pre-denaturation at 96 ℃ for 2 min;
96 ℃ for 6 seconds → 60 ℃ for 15 seconds, for a total of 45 cycles.
The primer and the probe for detecting the mycoplasma genitalium comprise a primer pair of a nucleotide sequence shown by SEQ ID NO 1-2, a probe of a nucleotide sequence shown by SEQ ID NO 3 and a primer pair of a nucleotide sequence shown by SEQ ID NO 4-5 and a probe of a nucleotide sequence shown by SEQ ID NO 6. the kit adopts human β globin gene as an internal reference to monitor whether the sampling process is abnormal, combines specific primers and probes designed aiming at mycoplasma genitalium outer membrane protein B gene to ensure higher sensitivity, can detect pathogens as low as 50Copies/ml, has short detection time, can complete the whole amplification detection process within 45 minutes, has NO cross reaction with pathogens similar to mycoplasma pneumoniae, mycoplasma hominis and the like, has NO influence on the detection result by common treatment drugs such as doxycycline, azithromycin, moxifloxacin and levofloxacin, has stronger specificity, can detect a low-value sample by 100%, has accurate and reliable detection results, and can be applied to the detection of clinical mycoplasma genitalium.
Detailed Description
The invention discloses a primer, a probe, a kit and a detection method for detecting mycoplasma genitalium, and a person skilled in the art can realize the detection by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
The primer, the probe, the kit and the material or the reagent used in the detection method for detecting the mycoplasma genitalium provided by the invention are all commercially available.
The invention is further illustrated by the following examples:
example 1 primers and probes of the invention
The primer and probe sequences of the invention are as follows:
TABLE 1 primer and Probe sequences of the invention
Example 2 kit of the invention
The components of the kit of this example are shown in Table 2.
TABLE 2
Wherein the reaction solution 1 comprises a basic buffer solution, a cation, an enhancer and an enzyme, the basic buffer solution is a Tris-HCl buffer solution containing BSA, and the cation is Mn2+、K+Including one or more of glycerol, DMSO, Tween20, Tricine X-100, potassium acetate and betaine, and the enzyme is bifunctional enzyme and/or UNG enzyme
Example 3 method for detecting Mycoplasma genitalium Using the kit of the invention
(1) Sample preparation: extracting nucleic acid of a sample to be detected, preparing a negative quality control product and a positive quality control product at the same time,
(2) PCR amplification System:
TABLE 3 PCR amplification System
In the PCR amplification system, the concentrations of Primer 1-Primer 4 are 20-35 pmol/mul, the concentrations of Probe 1-Probe 2 are 10-20 pmol/mul, and the concentrations of all components in the reaction solution 1 are as follows: 5-10U/ul of bifunctional enzyme, 0.1-0.5U/ul of UNG enzyme, 20mM dNTP, 2.8% glycerol, 3.7% DMSO, 0.01% Tween20, 60mM Tricine X-100, 110mM potassium acetate, 0.5M betaine and basic buffer solution; the PCR reaction solution 2 contains 20-30 mM MnCl2，P300。
(3) And (3) PCR amplification detection:
the amplification system of Table 2 was tested on a computer with amplification program of 50 ℃ for 2min, 96 ℃ for 2min, (96 ℃ for 6s, 60 ℃ for 15s) x 45 cycles; FAM and ROX channel fluorescence were collected at each cycle.
(4) And (5) judging a result:
if the ROX Ct value is more than or equal to 40 or the ROXCt value is less than or equal to 40 and the FAM has no typical S-type amplification curve, the data is invalid and needs to be retested; if the ROX Ct value is less than or equal to 40, the FAM Ct value is less than or equal to 45 and the amplification curve is an S-shaped curve, determining that the sample to be detected is positive MG; if the ROXCt value is less than or equal to 40, the FAM Ct value is greater than or equal to 45 or the FAM has no Ct, the sample to be detected is MG negative.
20 samples with the lowest detection limit (MG concentration of 50Copies/ml) were tested using the kit of example 2 according to the method described above, and the test results are shown in Table 4.
TABLE 4 Ct values for lowest detection Limit samples
The result shows that the detection result of the kit disclosed by the invention on 20 samples with the lowest detection limit is positive, and the accuracy can reach 100%.
EXAMPLE 4 comparison of the kits of the invention with commercially available reagents
The standard strain was diluted to the detection limit concentration (MG concentration: 50Copies/ml) and a total of 40 samples were tested in parallel with the commercial kit using the kit of example 2. The commercially available kit is a PCR-fluorescence-based Mycoplasma Genitalium (MG) nucleic acid detection kit which is purchased from Soviet biosciences, Inc. FAM Ct values results are shown in Table 5.
TABLE 5
The result shows that the detection results of the kit disclosed by the invention on 20 parts of the lowest detection limit samples are positive, and the detection results of the commercially available kit on 20 parts of the lowest detection limit samples are as follows: 9 positive results show that the sensitivity of the kit is higher than that of the commercially available kit.
Example 5
Control kit: specific primers and probes: primers 5-6, Probe3, internal standard Primer and Probe are the same as example 1, see primers 3-4 and Probe2 in Table 1; other components in the kit were the same as those in the kit of example 3
Wherein, the specific primers and the probes are as follows:
Primer5：5’-TGAGCCTTTCTAACCGCTGC-3’；
Prinmer6:5’-AAGCAAAGTTGCTCACGCTAC-3’；
Probe3:FAM-TGGGGTTATAACAGGTGTAGGTGGT–BHQ1；
20 samples with the lowest detection limit (MG concentration 50Copies/ml MG) were tested using example 3 and the control kit, and the results of FAMCt values are shown in Table 6.
TABLE 6
The result shows that the detection rate of the primer probe combination of the invention on the low-value sample is 100 percent, and the detection rate of the control kit on the low-value sample is only 65 percent (13/20), which indicates that the detection rate of the primer probe combination of the invention on the low-value sample is obviously superior to that of the primers and the probes of the control kit, and the detection result is more accurate and reliable.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Zhengzhou Antu bioengineering GmbH
<120> primer, probe, kit and detection method for detecting mycoplasma genitalium
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Claims (10)
1. A primer and probe for detecting mycoplasma genitalium, comprising:
a primer pair of a nucleotide sequence shown in SEQ ID NO. 1-2 and a probe of a nucleotide sequence shown in SEQ ID NO. 3.
2. The primer and probe according to claim 1, wherein the probe having the nucleotide sequence of SEQ ID NO. 3 has a fluorescence reporter FAM labeled at the 5 'end and a fluorescence quencher BHQ1 labeled at the 3' end.
3. A kit for detecting Mycoplasma genitalium, comprising the primer and the probe according to claim 1 or 2.
4. The kit according to claim 3, further comprising a primer for detecting the nucleotide sequence shown in SEQ ID NO. 4-5 of the reference gene and a probe for detecting the nucleotide sequence shown in SEQ ID NO. 6.
5. The kit needle according to claim 4, wherein the probe having the nucleotide sequence shown in SEQ ID NO. 6 has a fluorescence reporter ROX labeled at the 5 'end and a fluorescence quencher BHQ2 labeled at the 3' end.
6. The kit according to claim 3, further comprising a reaction solution 1 and a reaction solution 2; the reaction solution 1 comprises a basic buffer solution, cations, an enhancer and enzymes; the reaction solution 2 comprises ProClin300 and MnCl2。
7. The kit according to claim 3, wherein the base buffer is a Tris-HCl buffer containing BSA, and the cation is Mn2+、K+Including one or more of glycerol, DMSO, Tween20, Tricine X-100, potassium acetate, and betaine, the enzyme being a bifunctional DNA polymerase and/or a UNG enzyme.
8. The kit of claim 3, further comprising a negative quality control material and a positive quality control material, wherein the negative quality control material is a plasmid containing a human β globin gene fragment, and the positive quality control material comprises a plasmid containing an MG outer membrane protein B gene fragment and a plasmid containing a human β globin gene fragment.
9. A method for detecting mycoplasma genitalium, comprising: the kit of any one of claims 3 to 8, wherein the kit is used for real-time fluorescent quantitative PCR detection of the DNA of a sample to be detected, and the kit is positive when an S-shaped amplification curve is generated.
10. The detection method as claimed in claim 9, wherein the real-time fluorescence quantitative PCR system comprises:
the total volume is 50 mu L;
the real-time fluorescent quantitative PCR program comprises:
2 minutes at 50 ℃;
pre-denaturation at 96 ℃ for 2 min;
96 ℃ for 6 seconds → 60 ℃ for 15 seconds, for a total of 45 cycles.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111925941A (en) * | 2020-09-24 | 2020-11-13 | 北京健为医学检验实验室有限公司 | Virus preserving fluid and application thereof |
| CN112048566A (en) * | 2020-09-29 | 2020-12-08 | 山东绿都生物科技有限公司 | Product, method and application for simultaneously detecting mycoplasma gallisepticum and mycoplasma synoviae |
| CN113564233A (en) * | 2021-08-04 | 2021-10-29 | 杭州浙大迪迅生物基因工程有限公司 | Primer probe set and kit for human beta-tryptase mRNA RT-PCR detection |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108203738A (en) * | 2018-03-26 | 2018-06-26 | 郑州安图生物工程股份有限公司 | A kind of kit for being used to detect 1 type of human T-cell lymphotropic virus |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108203738A (en) * | 2018-03-26 | 2018-06-26 | 郑州安图生物工程股份有限公司 | A kind of kit for being used to detect 1 type of human T-cell lymphotropic virus |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111925941A (en) * | 2020-09-24 | 2020-11-13 | 北京健为医学检验实验室有限公司 | Virus preserving fluid and application thereof |
| CN111925941B (en) * | 2020-09-24 | 2021-02-05 | 北京健为医学检验实验室有限公司 | Virus preserving fluid and application thereof |
| CN112048566A (en) * | 2020-09-29 | 2020-12-08 | 山东绿都生物科技有限公司 | Product, method and application for simultaneously detecting mycoplasma gallisepticum and mycoplasma synoviae |
| CN113564233A (en) * | 2021-08-04 | 2021-10-29 | 杭州浙大迪迅生物基因工程有限公司 | Primer probe set and kit for human beta-tryptase mRNA RT-PCR detection |
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