CN117867146A - Kit, method and application for simultaneously detecting six pathogens - Google Patents
Kit, method and application for simultaneously detecting six pathogens Download PDFInfo
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- CN117867146A CN117867146A CN202311793548.1A CN202311793548A CN117867146A CN 117867146 A CN117867146 A CN 117867146A CN 202311793548 A CN202311793548 A CN 202311793548A CN 117867146 A CN117867146 A CN 117867146A
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- 238000000034 method Methods 0.000 title abstract description 15
- 239000000523 sample Substances 0.000 claims abstract description 68
- 238000001514 detection method Methods 0.000 claims abstract description 31
- 241000606153 Chlamydia trachomatis Species 0.000 claims abstract description 21
- 241000588652 Neisseria gonorrhoeae Species 0.000 claims abstract description 21
- 241000700584 Simplexvirus Species 0.000 claims abstract description 21
- 229940038705 chlamydia trachomatis Drugs 0.000 claims abstract description 21
- 241000204051 Mycoplasma genitalium Species 0.000 claims abstract description 18
- 241000204048 Mycoplasma hominis Species 0.000 claims abstract description 17
- 208000019802 Sexually transmitted disease Diseases 0.000 claims abstract description 9
- 241000202921 Ureaplasma urealyticum Species 0.000 claims abstract description 9
- 238000006243 chemical reaction Methods 0.000 claims description 41
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- 241000204003 Mycoplasmatales Species 0.000 claims description 6
- 239000003085 diluting agent Substances 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a kit, a method and application for simultaneously detecting six pathogens, wherein the six pathogens comprise chlamydia trachomatis, neisseria gonorrhoeae, ureaplasma urealyticum, mycoplasma genitalium, mycoplasma hominis and herpes simplex virus, and the kit comprises a forward primer, a reverse primer and a probe for detecting the six pathogens. The multiplex PCR detection technology based on the fluorescent probe can realize the simultaneous detection of six pathogen nucleic acids of chlamydia trachomatis, neisseria gonorrhoeae, ureaplasma urealyticum, mycoplasma genitalium, mycoplasma hominis and herpes simplex virus of a single sample, thereby accurately locking single or multiple pathogen infection types, having wide detection range and simultaneously being quick and accurate, further providing accurate information for treatment, and solving the problems of long culture period, poor antigen method sensitivity and low single detection coverage of nucleic acid under the condition of multiple infection in the current sexually transmitted disease pathogen detection method.
Description
Technical Field
The invention belongs to the technical field of nucleic acid amplification, and particularly relates to a kit, a method and application for simultaneously detecting six pathogens.
Background
Sexually transmitted diseases (sexually transmitted diseases, STD) refer to a group of infectious diseases transmitted through sexual or similar behavior. The important monitoring in China includes syphilis, AIDS, gonorrhea, genital herpes, condyloma acuminatum, nongonococcal urethritis (NGU) of chancre and lymphogranuloma venereal. The most common sexually transmitted diseases in China are nongonococcal urethritis, and common pathogens are Chlamydia Trachomatis (CT), ureaplasma Urealyticum (UU), mycoplasma Genitalium (MG), trichomonas vaginalis, candida albicans, herpesvirus and the like. Mycoplasma Hominis (MH) is one of the common pathogens of female genitourinary tract infection, and can cause genital tract diseases such as pelvic inflammatory disease, pyelonephritis, puerperal fever, colpitis and the like, and can also cause genital tract external infection. Genital herpes is a chronic life-long viral infectious disease caused by Herpes Simplex Virus (HSV) infection, type I (HSV-1) mainly invades the parts of oropharynx, tonsils, eyes, skin and the like, type II (HSV-2) mainly invades the parts of genitals, genital herpes can be repeatedly started, the health and psychological influence on patients is large, newborns can be infected through placenta and birth canal, and congenital infection of newborns is caused. Neisseria Gonorrhoeae (NG) can cause suppurative infections of the genitourinary system (gonorrhea), and is also one of the common pathogens of sexually transmitted diseases.
At present, detection of sexually transmitted disease pathogens mainly comprises smear microscopy, culture method, colloidal gold and the like, but the detection methods have the conditions of long culture method period, poor antigen method sensitivity, low single-detection coverage of nucleic acid under multiple infection and easy omission. The nucleic acid detection method is one of the common detection methods at present, and the multiplex PCR detection technology based on the fluorescent probe can realize the simultaneous detection of six pathogen nucleic acids of Chlamydia Trachomatis (CT), neisseria Gonorrhoeae (NG), ureaplasma Urealyticum (UU), mycoplasma Genitalium (MG), mycoplasma Hominis (MH) and Herpes Simplex Virus (HSV) of a single sample, thereby accurately locking single or multiple pathogen infection types and providing accurate information for treatment.
Disclosure of Invention
The invention provides a kit for simultaneously detecting six pathogens, which solves the problems of long culture period, poor antigen method sensitivity, low single-detection coverage of nucleic acid and easy omission of detection in the case of multiple infection in the conventional sexually transmitted disease pathogen detection method.
The technical scheme adopted by the invention for achieving the purpose is as follows: a kit for simultaneous detection of six pathogens, including chlamydia trachomatis, neisseria gonorrhoeae, mycoplasma urealyticum, mycoplasma genitalium, mycoplasma hominis, and herpes simplex virus, comprising forward primers, reverse primers, and probes for detection of the six pathogens.
Further, the primer pair and probe of the kit are as follows:
the forward primer and the reverse primer of the chlamydia trachomatis are respectively shown in SEQ ID NO.1 and SEQ ID NO.2, and the probe sequence is shown in SEQ ID NO. 3;
the forward primer and the reverse primer of the neisseria gonorrhoeae are respectively shown in SEQ ID NO.4 and SEQ ID NO.5, and the probe sequence is shown in SEQ ID NO. 6;
the forward primer and the reverse primer of the mycoplasma urealyticum are respectively shown in SEQ ID NO.7 and SEQ ID NO.8, and the probe sequence is shown in SEQ ID NO. 9;
the sequence of the forward primer and the reverse primer of the mycoplasma genitalium is shown as SEQ ID NO.10 and SEQ ID NO.11 respectively, and the sequence of the probe is shown as SEQ ID NO. 12;
the forward primer and the reverse primer of the human mycoplasma are respectively shown as SEQ ID NO.13 and SEQ ID NO.14, and the probe sequence is shown as SEQ ID NO. 15;
the sequences of the forward primer and the reverse primer of the herpes simplex virus are respectively shown as SEQ ID NO.16 and SEQ ID NO.17, and the sequences of the probes are shown as SEQ ID NO. 18;
further, two ends of the probe are respectively provided with a fluorescent group and a quenching group, wherein the fluorescent group is one of FAM, CY5, ROX and VIC, and the quenching group is one of BHQ1, BHQ2 and BHQ 3.
Further, the kit comprises an internal reference, the sequences of a forward primer and a reverse primer of the internal reference are respectively shown as SEQ ID NO.19 and SEQ ID NO.20, the sequences of a probe are respectively shown as SEQ ID NO.21, two ends of the probe are respectively provided with a fluorescent group and a quenching group, the fluorescent group is one of FAM, CY5, ROX and VIC, and the quenching group is one of BHQ1, BHQ2 and BHQ 3.
Further, the kit also comprises a PCR reaction solution, wherein the reaction solution comprises a reaction solution A, a reaction solution B and a reaction solution C 1 And reaction solution C 2 Wherein the reaction solution A comprises 1.5-5mM of Mg 2+ 0.1-0.5mM dNTP, 10 XPCR Buffer and enzyme dilution; the reaction solution B comprises 0.15-0.2U/. Mu.L of Taq enzyme, 0.04U/. Mu.L of UNG enzyme and enzyme diluent; the reaction solution C 1 Including primers and probes for Chlamydia trachomatis, primers and probes for Neisseria gonorrhoeae, primers and probes for Mycoplasma urealyticum, and primers and probes for internal references; the reaction solution C 2 Including primers and probes of mycoplasma genitalium, primers and probes of mycoplasma hominis, primers and probes of herpes simplex virus, and primers and probes of internal reference; wherein the reaction solution C 1 And reaction solution C 2 The primer concentrations were 0.5. Mu.M and the probe concentrations were 0.25. Mu.M.
Further, the kit also comprises a positive control and a negative control, wherein the positive control is pseudovirus and internal reference pseudovirus of a target amplification sequence, and the negative control is cell preservation solution and internal reference pseudovirus.
The invention also discloses a real-time fluorescence quantitative detection method for detecting sexually transmitted disease pathogens, which comprises the step of adding the primer pair and the probe combination of any one of claims 1-3 into a reaction system to perform real-time fluorescence quantitative PCR amplification.
The invention also discloses application of the kit, which is used for detecting one or more pathogens of chlamydia trachomatis, neisseria gonorrhoeae, ureaplasma urealyticum, mycoplasma genitalium, mycoplasma hominis and herpes simplex virus.
Compared with the prior art, the invention has the following advantages:
the fluorescent probe-based multiplex PCR detection technology can realize simultaneous detection of six pathogen nucleic acids of chlamydia trachomatis, neisseria gonorrhoeae, ureaplasma urealyticum, mycoplasma genitalium, mycoplasma hominis and herpes simplex virus of a single sample, thereby accurately locking single or multiple pathogen infection types, having wide detection range and simultaneously being rapid and accurate, and further providing accurate information for treatment;
secondly, the invention selects the human beta-actin as an internal reference, participates in whole-process extraction monitoring, controls false negative, has accurate amplification effect, high specificity and good sensitivity.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is an amplification plot of CT;
FIG. 2 is an amplification plot of NG;
FIG. 3 is a graph of amplification of UU;
FIG. 4 shows a reaction solution C 1 Amplification plot of IC;
FIG. 5 is an amplification plot of MH;
FIG. 6 is an amplification plot of MG;
FIG. 7 is a graph of HSV amplification;
FIG. 8 shows a reaction solution C 2 Amplification plot of IC;
FIG. 9 is a sensitivity verification graph of CT;
FIG. 10 is a sensitivity verification graph of NG;
fig. 11 is a sensitivity verification diagram of UU;
fig. 12 is a sensitivity verification diagram of MH;
FIG. 13 is a sensitivity verification graph of HSV;
fig. 14 is a sensitivity verification diagram of the MG.
Detailed Description
Embodiments of the present application are described in detail below with reference to the accompanying drawings.
Other advantages and effects of the present application will become apparent to those skilled in the art from the present disclosure, when the following description of the embodiments is taken in conjunction with the accompanying drawings. It will be apparent that the described embodiments are only some, but not all, of the embodiments of the present application. The present application may be embodied or carried out in other specific embodiments, and the details of the present application may be modified or changed from various points of view and applications without departing from the spirit of the present application. It should be noted that the following embodiments and features in the embodiments may be combined with each other without conflict. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, are intended to be within the scope of the present application.
It is noted that various aspects of the embodiments are described below within the scope of the following claims. It should be apparent that the aspects described herein may be embodied in a wide variety of forms and that any specific structure and/or function described herein is merely illustrative. Based on the present application, one skilled in the art will appreciate that one aspect described herein may be implemented independently of any other aspect, and that two or more of these aspects may be combined in various ways. For example, apparatus may be implemented and/or methods practiced using any number and aspects set forth herein. In addition, such apparatus may be implemented and/or such methods practiced using other structure and/or functionality in addition to one or more of the aspects set forth herein.
Example 1 design and Synthesis of primers and probes
Primers and probes for the six pathogens were designed based on NCBI published gene sequences of Chlamydia trachomatis, neisseria gonorrhoeae, mycoplasma urealyticum, mycoplasma genitalium, mycoplasma hominus and herpes simplex virus, and Primer5 was used for designing and optimizing the primers and probes for the six pathogens and internal reference, the Primer sequences and probe sequences involved are shown in Table 1. Primers and probes were synthesized by Shanghai Bai Ge Biotechnology Co.
TABLE 1 primer sequences and probe sequence Listing
Wherein both ends of the pathogen and the reference probe are respectively provided with a fluorescent group and a quenching group, the fluorescent group of the probe of the chlamydia trachomatis is FAM, the quenching group is BHQ1, the fluorescent group of the probe of the neisseria gonorrhoeae is CY5, the quenching group is BHQ3, the fluorescent group of the probe of the ureaplasma urealyticum is ROX, the quenching group is BHQ2, the fluorescent group of the probe of the mycoplasma genitalium is ROX, the quenching group is BHQ2, the fluorescent group of the probe of the mycoplasma hominis is FAM, the quenching group is BHQ1, the fluorescent group of the probe of the herpes simplex virus is CY5, the quenching group is BHQ3, the fluorescent group of the probe of the reference probe is VIC, and the quenching group is BHQ1.
Example 2 methods of Using the kit
S1: sampling
Extracting pseudoviruses or clinical samples by using a nucleic acid extraction reagent of Tianlong Ex-DNA/RNA virus 4.0 pre-sealing plate batch number 21022020T324 according to a using method of an extraction instruction to obtain corresponding nucleic acid extraction products;
s2: PCR amplification
(1) Thawing the required components in the kit, reversing, uniformly mixing and centrifuging for later use; primers and probes corresponding to each pathogen were mixed with sterile, nuclease-free double distilled water, and PCR reaction solutions were prepared according to table 2.
TABLE 2 composition of PCR reaction solution
| Reagent name | Component (A) | Addition amount/. Mu.L |
| Reaction solution A | Mg 2+ dNTP, 10 XPCR Buffer, enzyme dilution | 6 |
| Reaction liquid B | Taq enzyme, UNG enzyme and enzyme diluent | 8 |
| Reaction solution C 1 | CT, NG, UU, IC primer and probe | 6 |
| Reaction solution C 2 | MG, MH, HSV, IC primer and probe | 6 |
The detection method of the invention is that two pipes are combined, and the pipe 1 is the reaction liquid A+the reaction liquid B+the reaction liquid C 1 Tube 2 is reaction solution A+reaction solution B+reaction solution C 2 The total reaction volume of each tube was 20. Mu.L, and the nucleic acid extraction product was 20. Mu.L.
Wherein, the concentration of each primer is 0.5 mu M, the concentration of each probe is 0.25 mu M, and the concentration of Mg 2+ The working concentration of (2) was 5mM, the working concentration of dNTP was 0.3mM, the working concentration of Taq enzyme was 0.2U/. Mu.L, and the working concentration of UNG enzyme was 0.04U/. Mu.L. In addition, the kit also comprises a positive reference substance and a negative reference substance, wherein the positive reference substance is the false of the target amplified sequenceThe negative control is cell preservation solution and internal reference pseudovirus.
(2) Adding 20 mu L of templates into the prepared reaction system, covering a tube cover, shaking, mixing uniformly and performing instantaneous centrifugation, wherein the templates added into the tube 1 are chlamydia trachomatis, neisseria gonorrhoeae and ureaplasma urealyticum, the templates added into the tube 2 are mycoplasma hominis, herpes simplex virus, mycoplasma genitalium and internal references, the detection channels are FAM, CY5, ROX and VIC, and the reaction tubes are sequentially placed into a PCR instrument for amplification reaction according to the conditions shown in Table 3:
TABLE 3 amplification conditions
Setting the fluorescent internal reference of the instrument as "None", editing the sample information of each reaction well according to the instrument operation rules, and selecting a corresponding detection target.
(3) Analysis of results
Setting a base line according to specific amplification conditions of different instruments and samples, wherein a threshold line is selected above a negative control, and the result judgment standard is as follows: detecting that the target has no numerical value, wherein a sample with an internal standard Ct value smaller than 40 is non-reactive; the internal standard has no numerical value and is invalid, and resampling or extraction amplification is needed; the sample with Ct value smaller than 40 is reactivity, and specific judgment standards and methods are shown in Table 4.
TABLE 4 determination criteria for detection results
Example 3 specificity and sensitivity experiments
The kit and the method are used for detecting clinical negative samples and non-target (six) pseudoviruses, the specific detection results are shown in figures 1-8, the results indicate that positive pseudovirus signals corresponding to amplification are S-shaped curves, amplified NC negative samples and other pseudoviruses are negative, and the specificity of the primer probes and the kit of the method is good.
The pseudoviruses with the determined values were diluted to 300copy/mL with physiological saline, and extracted and amplified as in example 2. The detection results are shown in figures 9-14, and the results show that the detection of 20 multiple holes can meet the detection requirement of more than 19, which indicates that the kit has higher sensitivity.
The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be included within the scope of the present invention.
Claims (8)
1. A kit for simultaneous detection of six pathogens, characterized in that: the six pathogens comprise chlamydia trachomatis, neisseria gonorrhoeae, ureaplasma urealyticum, mycoplasma genitalium, mycoplasma hominis and herpes simplex virus, and the kit comprises a forward primer, a reverse primer and a probe for detecting the six pathogens.
2. The kit of claim 1, wherein:
the forward primer and the reverse primer of the chlamydia trachomatis are respectively shown in SEQ ID NO.1 and SEQ ID NO.2, and the probe sequence is shown in SEQ ID NO. 3;
the forward primer and the reverse primer of the neisseria gonorrhoeae are respectively shown in SEQ ID NO.4 and SEQ ID NO.5, and the probe sequence is shown in SEQ ID NO. 6;
the forward primer and the reverse primer of the mycoplasma urealyticum are respectively shown in SEQ ID NO.7 and SEQ ID NO.8, and the probe sequence is shown in SEQ ID NO. 9;
the sequence of the forward primer and the reverse primer of the mycoplasma genitalium is shown as SEQ ID NO.10 and SEQ ID NO.11 respectively, and the sequence of the probe is shown as SEQ ID NO. 12;
the forward primer and the reverse primer of the human mycoplasma are respectively shown as SEQ ID NO.13 and SEQ ID NO.14, and the probe sequence is shown as SEQ ID NO. 15;
the sequences of the forward primer and the reverse primer of the herpes simplex virus are respectively shown as SEQ ID NO.16 and SEQ ID NO.17, and the sequences of the probes are shown as SEQ ID NO. 18.
3. The kit of claim 2, wherein: the two ends of the probe are respectively provided with a fluorescent group and a quenching group, wherein the fluorescent group is one of FAM, CY5, ROX and VIC, and the quenching group is one of BHQ1, BHQ2 and BHQ 3.
4. A kit according to any one of claims 1 to 3, wherein: the kit comprises an internal reference, wherein the sequences of a forward primer and a reverse primer of the internal reference are respectively shown as SEQ ID NO.19 and SEQ ID NO.20, the sequence of a probe is shown as SEQ ID NO.21, two ends of the probe are respectively provided with a fluorescent group and a quenching group, the fluorescent group is one of FAM, CY5, ROX and VIC, and the quenching group is one of BHQ1, BHQ2 and BHQ 3.
5. The kit of any one of claims 1-4, wherein: the kit also comprises a PCR reaction solution, wherein the reaction solution comprises a reaction solution A, a reaction solution B and a reaction solution C 1 And reaction solution C 2 Wherein the reaction solution A comprises 1.5-5mM of Mg 2+ 0.1-0.5mM dNTP, 10 XPCR Buffer and enzyme dilution; the reaction solution B comprises 0.15-0.2U/. Mu.L of Taq enzyme, 0.04U/. Mu.L of UNG enzyme and enzyme diluent; the reaction solution C 1 Including primers and probes for Chlamydia trachomatis, primers and probes for Neisseria gonorrhoeae, primers and probes for Mycoplasma urealyticum, and primers and probes for internal references; the reaction solution C 2 Including primers and probes of mycoplasma genitalium, primers and probes of mycoplasma hominis, primers and probes of herpes simplex virus, and primers and probes of internal reference; wherein the reaction solution C 1 And reaction solution C 2 The primer concentrations were 0.5. Mu.M and the probe concentrations were 0.25. Mu.M.
6. The kit of any one of claims 1-5, wherein: the kit also comprises a positive reference substance and a negative reference substance, wherein the positive reference substance is pseudovirus and internal reference pseudovirus of the target amplification sequence, and the negative reference substance is cell preservation solution and internal reference pseudovirus.
7. A real-time fluorescent quantitative detection method for detecting sexually transmitted disease pathogens is characterized in that: comprising the step of adding the primer pair and probe combination of any one of claims 1 to 3 to a reaction system for real-time fluorescent quantitative PCR amplification.
8. A kit according to any one of claims 1 to 6 for the detection of one or more pathogens from chlamydia trachomatis, neisseria gonorrhoeae, mycoplasma urealytium, mycoplasma genitalium, mycoplasma hominis and herpes simplex virus.
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| CN202311793548.1A CN117867146A (en) | 2023-12-25 | 2023-12-25 | Kit, method and application for simultaneously detecting six pathogens |
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| CN202311793548.1A CN117867146A (en) | 2023-12-25 | 2023-12-25 | Kit, method and application for simultaneously detecting six pathogens |
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