CN117737306A - Compositions, kits and methods for detecting sexually transmitted infectious pathogens - Google Patents
Compositions, kits and methods for detecting sexually transmitted infectious pathogens Download PDFInfo
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Abstract
The invention discloses a composition, a kit and a detection method for detecting a sexually transmitted infectious pathogen, wherein the composition comprises at least one of a component A for detecting human immunodeficiency virus type I, a component B for detecting treponema pallidum and a component C for detecting monkey pox virus, the component A comprises a nucleotide sequence shown as SEQ ID NO 1-3, the component B comprises a nucleotide sequence shown as SEQ ID NO 4-6, and the component C comprises a nucleotide sequence shown as SEQ ID NO 7-9. The composition provided by the invention can detect 3 sexually transmitted infectious pathogens, has high sensitivity, good precision, high specificity and strong anti-interference performance, and has good application prospect in the field of detection of sexually transmitted infectious pathogens.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a composition, a kit and a detection method for detecting a detectably transmitted infectious pathogen.
Background
Sexually transmitted infections (Sexually transmitted infections, STI) are an important concept in comprehensive education, meaning infections that occur primarily through sexual contact behavior, and also infections caused by indirect contact transmission. Sexually transmitted diseases are caused by sexually transmitted infections, and common sexually transmitted diseases include syphilis, gonorrhea, genital herpes, condyloma acuminatum, nongonococcal urethritis, venereal lymphogranuloma, AIDS, and the like. And timely cutting off sexually transmitted infection pathways, performing consultation and behavioral intervention, timely detection treatment after high risk behaviors, vaccinating or receiving other biomedical interventions can prevent sexually transmitted infections.
Monkey pox is a viral zoonotic disease, caused by the monkey pox virus, which can be transmitted between animals and humans, and can also be transmitted secondarily between humans. The monkey poxvirus is an enveloped double-stranded DNA virus which is rectangular and can be cultivated and grown in African green monkey kidney cells to cause cytopathy, belongs to orthopoxvirus genus of poxviridae, resists diethyl ether, has strong resistance to dryness, and is easy to be inactivated by chloroform, methanol and formalin. Heating at 56 ℃ for 30 minutes also easily deactivates it.
The human immunodeficiency virus has a diameter of about 80-140 nanometers and is round or oval. The outer membrane of the virus is a lipid envelope, derived from a host cell, and embedded with the viral proteins gp120 and gp41. Wherein gp41 is a transmembrane protein and gp120 is located on the surface and is bound to gp41 by non-covalent interactions. Inward is a spherical matrix (matrix) formed by protein p17, and a semi-conical capsid (capsid) formed by protein p24, which is of high electron density under electron microscopy. The capsid contains the viral RNA genome, enzymes (reverse transcriptase, integrase, protease) and other components from the host cell (e.g. tRNAlys3 as primers for reverse transcription). The HIV detection is a key component of the HIV prevention and treatment program, and is also a high-benefit HIV prevention and intervention measure.
Syphilis is a chronic infectious disease, which is a systemic infection at the beginning and has slow disease course, and invades various organs and tissues of human body in the development process, and can also be hidden for years and even has no clinical manifestation for the whole life. Syphilis can be classified into acquired syphilis (acquired) and congenital syphilis from infectious sources. Acquired syphilis manifests itself in time during the long-term course of disease due to changes in body resistance and reactivity.
Existing detection tools capable of accurately detecting sexually transmitted infectious agents tend to be expensive and not readily available. Furthermore, the time required to receive the test results is often long, which may affect follow-up, resulting in incomplete care or treatment.
There is therefore a great need in the art for a simple, rapid detection of sexually transmitted infectious agents that achieves high sensitivity, high specificity and robust detection of sexually transmitted infectious agents.
Disclosure of Invention
The invention aims to provide a composition, a kit and a detection method for detecting sexually transmitted infectious pathogens, so as to realize detection with high sensitivity, high specificity and strong anti-interference performance on sexually transmitted infectious pathogens.
The present invention provides a composition for detecting a sexually transmitted infectious pathogen comprising at least one of component a, component B, component C:
the component A comprises a nucleotide sequence shown as SEQ ID NO. 1-3, and is used for detecting human immunodeficiency virus type I (HIV-1);
the component B comprises a nucleotide sequence shown as SEQ ID NO. 4-6, and is used for detecting Treponema Pallidum (TP);
the component C comprises nucleotide sequences shown in SEQ ID NO. 7-9, and is used for detecting monkey pox virus (MPXV).
The composition for detecting sexually transmitted infectious pathogens provided by the invention can detect and distinguish 3 sexually transmitted infectious pathogens (human immunodeficiency virus type I, treponema pallidum and monkey poxvirus) through careful design of nucleotide sequences (upstream and downstream primers and corresponding probes), has high sensitivity, good precision, high specificity and strong anti-interference performance, and simultaneously improves detection efficiency, shortens detection period, reduces detection cost, provides more sufficient and rapid diagnosis for clinic and basis for eliminating different pathogen infections, shortens time of clinical diagnosis, quickens implementation of treatment measures, and has good application prospect in the field of detection of sexually transmitted infectious pathogens.
Preferably, the composition for detecting a sexually transmitted infectious pathogen further comprises a nucleotide sequence for detecting an internal standard.
Preferably, the nucleotide sequence for detecting the internal standard is shown as SEQ ID NO. 10-12, and/or the internal standard comprises the nucleotide sequence shown as SEQ ID NO. 28.
Preferably, the nucleotide sequences of the component A, the component B, the component C and the internal standard for detection comprise probes, and fluorescent reporter groups are marked on the probes.
Preferably, the components of the composition for detecting a sexually transmitted infectious pathogen are present in a mixed form.
Preferably, the composition for detecting a sexually transmitted infectious pathogen comprises component a, component B and component C.
The present invention provides a kit for detecting a sexually transmitted infectious pathogen, comprising the composition for detecting a sexually transmitted infectious pathogen.
Preferably, the kit for detecting a sexually transmitted infectious pathogen further comprises a nucleic acid releasing reagent, a nucleic acid extracting reagent, a reverse transcriptase, a DNA polymerase, dNTP mix, dUTP, UNG enzyme, PCR buffer, and Mg 2+ At least one of them.
Preferably, the kit for detecting a sexually transmitted infectious pathogen further comprises at least one of a positive quality control, a negative quality control.
The composition for detecting sexually transmitted infectious pathogens is used for constructing a kit for detecting sexually transmitted infectious pathogens, so that the sexually transmitted infectious pathogens can be accurately detected, and the high specificity, the high sensitivity, the high anti-interference performance and the high precision are achieved.
The present invention also provides a method for detecting a sexually transmitted infectious pathogen for non-diagnostic purposes, comprising the steps of:
s1, extracting nucleic acid of a sample to be detected to obtain a nucleic acid sample;
s2, performing PCR amplification on the nucleic acid sample by using the kit for detecting the detectably transmitted infectious pathogen;
s3, obtaining and analyzing a result.
The kit for detecting sexually transmitted infectious pathogens provided by the invention is simple in operation and rapid in detection when detecting sexually transmitted infectious pathogens, and can achieve higher accuracy and sensitivity.
Drawings
FIG. 1 is a graph showing the amplification of human immunodeficiency virus type I, treponema pallidum, monkey pox virus and internal standard in example 1.
FIG. 2 is a diagram showing the specificity of the PCR reaction system of example 2.
FIG. 3 is a graph showing the detection result of the sensitivity of the PCR reaction system of example 3 at 500copies/mL for detecting human immunodeficiency type I virus.
FIG. 4 is a graph showing the detection result of the sensitivity of the PCR reaction system of example 3 at 500copies/mL for detecting treponema pallidum.
FIG. 5 is a graph showing the sensitivity of the PCR reaction system of example 3 at 500copies/mL for detection of monkey pox virus.
FIG. 6 is a graph showing the detection results of the PCR reaction system of example 4 for human immunodeficiency type I virus.
FIG. 7 is a graph showing the results of the detection of precision of the treponema pallidum by the PCR reaction system of example 4.
FIG. 8 is a graph showing the detection results of the PCR reaction system of example 4 on the precision of the detection of the monkey poxvirus.
FIG. 9 is a graph showing the results of the PCR reaction system of comparative example 1 with respect to human immunodeficiency type I virus
FIG. 10 is a graph showing the detection results of treponema pallidum by using the other different primer probes designed for the PCR reaction system of comparative example 1.
FIG. 11 is a graph showing the results of detection of monkey poxvirus by the remaining different primer probes designed for the PCR reaction system of comparative example 1.
FIG. 12 is a graph showing the results of the combined detection of human immunodeficiency virus type I, treponema pallidum and monkey pox virus with the other different primer probes designed for the PCR reaction system of comparative example 1.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution of the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments.
Example 1
1. Reagent preparation:
the PCR reaction solution is shown in table 1, the corresponding sequences of the upstream and downstream primers and probes are shown in table 2, and the upstream and downstream primers and probes provided in table 2 are the compositions for detecting the sexually transmitted infectious pathogens provided in this example.
The preparation of the enzyme mixture is as follows: neoscript RT reverse transcriptase and H-Taq enzyme (1. Mu.L RT enzyme at 5U/. Mu.L and 3. Mu.L H-Taq enzyme at 5U/. Mu.L per human)
According to the number of samples to be detected, positive controls (inactivated clinical samples positive for pathogens in three detection ranges) and negative controls (sterile physiological saline is taken as a matrix), corresponding amounts of PCR reaction liquid and enzyme mixed liquid are taken according to a proportion (36 mu L of PCR reaction liquid/human part + 4 mu L of enzyme mixed liquid/human part), and are fully and uniformly mixed into PCR mixed liquid, and centrifugation is carried out at 2000rpm for 10s for later use.
The differentiation of signals was performed for different sexually transmitted infectious pathogens (human immunodeficiency virus type I, treponema pallidum, monkey poxvirus) using different fluorescent reporter groups labeled on different probes, respectively. Wherein the fluorescence report reporter of the probe for detecting the human immunodeficiency virus I is CY5; the fluorescent reporter group of the probe for detecting treponema pallidum is ROX; the fluorescent reporter group of the probe for detecting the monkey poxvirus is FAM; the fluorescent reporter group of the probe for detecting the internal standard (the nucleotide sequence is shown as SEQ ID NO: 28) is HEX.
SEQ ID NO. 28 sequence:
TTCATACTCATGGCGAAGCACGCAACAAGCCGAACCCTCAATCCCAGTGAGGACTACTCCGTTGGGGGTTACTTCGCAAGGACGCCGCATGTGCAGTTCCTCAGTCTTCCTTCTCCTCCATATGAAGATTTATCCCATACGGTGCTAGCGAAGCAAGTCTTCCGAGTTCATACTGCATCTCGAACGAAGGTCACTCACAGTTCCGAAGGTCACTACGGTTTCCGAGTCTTATTGTTCTTCTTCTTCCACGTATGACAGATACCCTCAACCCTCAGGCCCACGGGGGGTTGCTACGCCCAGGGCTACTCCGTTGGCCCTGAAGGGTTACTTCGTTAGGGCTACTTGGAGGAGTTGACCAACCCGAACATGGTGCCTTGATCTTCAGCTTTCCAATAAGAAGATCATTGATGCAGCACACTGAGCTAAATTCTTAAACTATCTTTACCGGTGTGCTGCCTCGCAGAGATTCTCAACCTAGTCCGCGCATACTCTTTCCGTTTCTGTCTTACAGTCTTTCTTGTTCTTCTTTTCCCTATAGTTAGAAGATGAGCCCCTGGGGTGCCCTGCCCTTCTTTTACGGCTGATGGACATTCAATAGATTTATCTGCTGTTGCCTGATCTACGACCCCCTCAATCTGTCGAAGCAAATGCTGCACAGAGGGAGAAGTTGCCTGACCGGGCTATTCCCTTGTTATCTGTTGTCCAACTGGTTGAATAGTAATCTGTGCTGGATCCCTCTGAAGCTGGTCATTC
PCR (polymerase chain reaction) reaction systems include PCR buffers, DNA polymerases, dNTPs, primers and probes, and metal cations required to catalyze the DNA polymerase reaction. PCR buffer is made of Tris-HCl, KCl, naCl 2 These buffer systems were composed (purchased from Hunan Kangda Biotechnology Co., ltd.).
TABLE 1 composition of PCR reaction solution
The PCR reaction solution and the enzyme mixed solution prepared according to the table 1 are additionally matched with a negative control taking sterile physiological saline as a matrix, and the inactivated clinical samples positive to pathogens in three detection ranges are taken as positive controls to form the detection kit.
TABLE 2 primer probe sequences
2. Sample processing and sample addition
The treatment method comprises the following steps: nucleic acid extraction was performed using a nucleic acid extraction or purification reagent (S10016E) from Santa Clara Biotechnology Co., ltd according to the instructions to obtain a treated sample.
And 5 mu L of each of the treated sample, the negative control and the positive control are respectively sucked into corresponding 0.2mL PCR reaction tubes, 40 mu L of PCR mixed solution and 5 mu L of internal standard are added into each tube, and the tube cover is covered.
3. PCR amplification
And (3) performing PCR amplification according to a certain temperature and time setting program on the SLAN-96P full-automatic medical PCR analysis system. The preferred embodiment of the present invention is shown in Table 3, and the SLAN-96P fully automatic medical PCR analysis system is used in this example:
TABLE 3 Table 3
Enzyme activation in table 3: mainly activating Taq enzyme activity.
4. Detection of
(1) The criteria are shown in table 4:
TABLE 4 Table 4
(2) Detection result
The detection results are shown in FIG. 1, and the results in FIG. 1 show that the PCR mixture can carry out joint detection and distinction on human immunodeficiency virus type I (HIV-1), treponema Pallidum (TP) and monkey pox virus (MPXV).
Therefore, the composition for detecting sexually transmitted infectious pathogens provided by the invention can realize detection and distinction of 3 sexually transmitted infectious pathogens at the same time, and has high specificity and high sensitivity, and the upstream primer and the downstream primer corresponding to each sexually transmitted infectious pathogen have small interaction with the corresponding probe.
Example 2
Specific detection
Other sexually transmitted infectious pathogens were specifically detected using the composition for detecting sexually transmitted infectious pathogens provided in example 1, and the specific detection results are shown in fig. 2. As can be seen from FIG. 2, the composition for detecting sexually transmitted infectious pathogens provided in example 1 does not have nonspecific amplification and cross-reaction to other sexually transmitted infectious pathogens (hepatitis B virus, hepatitis C virus, EB virus, gonorrhea, chlamydia trachomatis, human papillomavirus). This demonstrates that the composition provided in example 1 for detecting sexually transmitted infectious pathogens is highly specific.
Example 3
Sensitivity detection
The results of sequential human immunodeficiency virus type I (HIV-1), treponema Pallidum (TP) and monkey pox virus (MPXV) sensitivity tests using the compositions provided in example 1 for detecting sexually transmitted infectious pathogens are shown in FIGS. 3-5. As can be seen from fig. 3 to 5, the composition for detecting sexually transmitted infectious pathogens provided by the present invention can still accurately detect each channel of the sample to be tested containing human immunodeficiency virus type I (HIV-1), treponema Pallidum (TP) and monkey pox virus (MPXV) at a concentration as low as 500.0copies/mL, thereby demonstrating that the sensitivity of the composition for detecting sexually transmitted infectious pathogens provided in example 1 is high.
Example 4
Precision detection
The embodiment selects medium and low concentrationLow concentration 2 sample concentration levels, concentrations 2.5X10 respectively 4 Copies/mL and 2500Copies/mL were measured for in-batch precision and inter-batch precision, and each sample was assayed in 6 replicates. The results show that the detection rates of the medium-low concentration sample and the low concentration sample are 100%, and the detection results of the precision reagent show that the composition for detecting the sexually transmitted infectious agents provided in the embodiment 1 has better reagent performance, and the precision CV values in the batch and the batch are far less than 5% index (see figures 6-8).
Example 5
Tamper resistance detection
The test kit for detecting a sexually transmitted pathogen provided in example 1 was analyzed for interferents by the method of EP7-A, clinical Biochemical interference assay, and the amplification results in the presence of interferents are shown in Table 5. As can be seen from Table 5, positive samples of 3 sexually transmitted infectious agents were all detected in the samples tested, and the PCR interfering substances of bilirubin (28 mg/dL), triglyceride (3000 mg/dL) and hemoglobin (2000 mg/dL) at a certain concentration had no significant effect on the composition for detecting sexually transmitted infectious agents provided in example 1. This demonstrates that the composition for detecting sexually transmitted infectious agents provided in example 1 has good specificity and no interference between sexually transmitted infectious agents.
TABLE 5
Comparative example 1
Comparative example 1 sexually transmitted infectious pathogens were detected with reference to example 1, and comparative example 1 differs from example 1 in that: other primers and probes for detecting sexually transmitted pathogens are used to compose different detection systems. In addition to the above differences, the other materials used in this comparative example and the process operation were exactly the same as in example 1. Comparative example 1 primer probe sequences are shown in table 6 below.
TABLE 6 comparative example 1 primer probe sequences
As shown in FIGS. 9-11, the single detection results in comparative example 1 show that the single detection results under different primer probes are better as shown in FIGS. 9-11. The best primer probes HIV-1 (SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17), TP (SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO: 22), MPXV (SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO: 27) were selected. As shown in fig. 12, the results of the joint inspection in comparative example 1 show that the system had a significant delamination of the amplification curve and a significant decrease in fluorescence increase after the joint inspection was completed as shown in fig. 12, and the overall detection effect was poor even for individual targets without amplification curve.
Therefore, in the invention, the combination of the primer probes is not replaceable and replaced, and other primers and probes are adopted to form different detection systems, and the detection system is also used for detecting 3 sexually transmitted infectious pathogens, so that the overall detection effect is poor. The composition for detecting sexually transmitted infectious pathogens provided by the invention can detect 3 sexually transmitted infectious pathogens simultaneously, and has excellent precision, specificity, sensitivity and anti-interference performance.
The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A composition for detecting a sexually transmitted infectious pathogen, comprising at least one of component a, component B, and component C:
the component A comprises a nucleotide sequence shown as SEQ ID NO. 1-3, and is used for detecting human immunodeficiency virus I;
the component B comprises a nucleotide sequence shown as SEQ ID NO. 4-6, and is used for detecting treponema pallidum;
the component C comprises nucleotide sequences shown in SEQ ID NO. 7-9, and is used for detecting the monkey pox virus.
2. The composition for detecting a sexually transmitted infectious pathogen according to claim 1, wherein the composition for detecting a sexually transmitted infectious pathogen further comprises a nucleotide sequence for detecting an internal standard.
3. The composition for detecting a sexually transmitted infectious pathogen according to claim 2, wherein the nucleotide sequence for detecting an internal standard is shown in SEQ ID NOs 10 to 12 and/or the internal standard comprises the nucleotide sequence shown in SEQ ID NO 28.
4. The composition for detecting a sexually transmitted infectious pathogen according to claim 1, wherein said component a, said component B, said component C and said nucleotide sequence for detecting an internal standard each comprise a probe, said probe having a fluorescent reporter group labeled thereon.
5. The composition for detecting a sexually transmitted infectious pathogen according to claim 1, wherein the components of the composition for detecting a sexually transmitted infectious pathogen are present in a mixed form.
6. The composition for detecting a sexually transmitted infectious pathogen of claim 1, comprising component a, component B, and component C.
7. A kit for detecting a sexually transmitted infectious pathogen, comprising a composition for detecting a sexually transmitted infectious pathogen according to any one of claims 1-6.
8. The kit for detecting a sexually transmitted infectious pathogen according to claim 7, further comprising a nucleic acid releasing reagent, a nucleic acid extracting reagent, a reverse transcriptase, a DNA polymerase, dNTP mix, dUTP, UNG enzyme, PCR buffer, and Mg 2+ At least one of them.
9. The kit for detecting a sexually transmitted infectious pathogen according to claim 7, wherein the kit for detecting a sexually transmitted infectious pathogen further includes at least one of a positive quality control and a negative quality control.
10. A method for detecting a sexually transmitted infectious agent for non-diagnostic purposes, comprising the steps of:
s1, extracting nucleic acid of a sample to be detected to obtain a nucleic acid sample;
s2, performing PCR amplification on the nucleic acid sample by using the kit for detecting the detectably transmitted infectious agent according to any one of claims 7 to 9;
s3, obtaining and analyzing a result.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311771264.2A CN117737306A (en) | 2023-12-21 | 2023-12-21 | Compositions, kits and methods for detecting sexually transmitted infectious pathogens |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311771264.2A CN117737306A (en) | 2023-12-21 | 2023-12-21 | Compositions, kits and methods for detecting sexually transmitted infectious pathogens |
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| CN117737306A true CN117737306A (en) | 2024-03-22 |
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| CN202311771264.2A Pending CN117737306A (en) | 2023-12-21 | 2023-12-21 | Compositions, kits and methods for detecting sexually transmitted infectious pathogens |
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