CN115976274A - Composition, kit, method and application for detecting pathogenesis cause of lichen planus - Google Patents
Composition, kit, method and application for detecting pathogenesis cause of lichen planus Download PDFInfo
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Abstract
The invention belongs to the field of molecular biological detection, and particularly relates to a composition for detecting pathogenesis causes of lichen planus, and more particularly relates to a composition, a kit, a method and application for detecting HHV-7, HPV-11 and HPV-16. The composition provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting different loci on different pathogens, thereby simultaneously realizing the detection and typing of three viruses in a single-tube reaction system. Allowing different pathogens to be treated differently, thus making treatment and prevention more effective. Meanwhile, the highest detection sensitivity of the composition can reach 200 copies/mL, and the detection is more accurate.
Description
Technical Field
The invention belongs to the field of molecular biological detection, and particularly relates to a composition for detecting pathogenesis causes of lichen planus, and more particularly relates to a composition, a kit, a method and application for detecting HHV-7, HPV-11 and HPV-16.
Background
Lichen Planus (LP), also known as lichen ruber planus, is a self-limiting disease with unclear etiology, and the exact etiology and pathogenesis of LP are not clear until now. With the rapid development of basic subjects such as immunology and molecular biology in recent years, the pathogenesis of LP is deeply understood. Research shows that cell-mediated immune response, keratinocyte apoptosis, change of cell differentiation marker molecules and neuropsychiatric factors are closely related to the incidence of lichen planus. At present, the pathogenesis of the virus infection participating in the lichen planus becomes a research hotspot at home and abroad.
HHV-7, which is latent in saliva or monocytes, is activated in LP patients to allow viral replication into skin tissue. HHV-7 can cause cytokine network imbalance after intracellular proliferation, leading to cytopathic effect and pathological change of LP, so that HHV-7 is one of pathogenic factors of LP.
Human papillomavirus is a DNA tumor virus. Its growth characteristics are epitheliophilic. In recent years, it has attracted attention as a possible cause of Oral Lichen Planus (OLP). The damage caused by HPV is an immune response in the cellular medium, consistent with the hypothesis that OLP pathogenesis is due to local autoimmune disorders. The research result shows that HPV-11 and 16 types of positive are respectively detected in the tissues of OLP patients, and the positive rate is as high as 86.7 percent.
In conclusion, although the pathogenesis of LP is not clear at present and the potential pathogen species are very complex, HHV-7, HPV-11 and HPV-16 are all pathogenic factors of LP, so that the demands exist for detecting the pathogens and establishing the pathogenic factors of patients so as to treat diseases and reduce the risk of HPV complications.
Disclosure of Invention
In view of the above, in a first aspect, the present invention provides a composition for detecting a predisposition for developing lichen planus. Simultaneously, the method comprises the following steps:
upstream and downstream primers and probes for detecting HHV-7 as shown in SEQ ID NO 1-3;
the upstream and downstream primers and probes for detecting HPV-11 as shown in SEQ ID NO 4-6; and
the upstream and downstream primers and probes for detecting HPV-16 are shown in SEQ ID NO 7-9.
Further, the composition comprises: and detecting upstream and downstream primers and probes of the internal standard.
In some specific embodiments, the internal standard is a human internal standard gene. In a specific embodiment, the internal standard is GAPDH.
In some specific embodiments, the upstream and downstream primers and probes for detecting the internal standard are the internal standard upstream primer shown in SEQ ID NO. 10, the internal standard downstream primer shown in SEQ ID NO. 11, and the internal standard probe shown in SEQ ID NO. 12.
The composition provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting different sites on the different pathogens, thereby simultaneously realizing the detection and typing of three viruses in a single-tube reaction system. Allowing different pathogens to be treated differently, thus making treatment and prevention more effective. Meanwhile, the highest detection sensitivity of the composition can reach 200 copies/mL, and the detection is more accurate.
Further, the composition further comprises: 31-33 shown in SEQ ID NO, and upstream and downstream primers and probes for detecting HHV-7.
The use of the above composition enables the simultaneous detection of two targets of HHV-7, avoiding the risk of missed detection due to mutation, reducing the probability of false negatives, further increasing the accuracy of the detection.
Further, the fluorescent groups of the probes of the composition of the present invention are different from each other and do not interfere with each other.
Herein, "different from each other and non-interfering" means that the fluorophores used in the composition for the probes to detect each target are different and do not interfere with each other's detection, i.e., can be detected using different channels. For example, FAM, HEX, ROX and CY5 can be used, which do not have close absorbance values and can select different channels and thus do not interfere with each other.
Further, in some embodiments, the compositions of the invention may include one or more of the primer and probe pairs described above. In the present invention, "pair" refers to the matched upstream and downstream primers and probes for detecting a target.
For example, any 4 pairs of the 5 pairs of primers and probes may be included, any 3 pairs of the 5 pairs of primers and probes may be included, any 2 pairs of the 5 pairs of primers and probes may be included, and any 1 pair of the 5 pairs of primers and probes may be included.
In some specific embodiments, the compositions of the invention are used in fluorescence PCR.
In a specific embodiment, the fluorescent reporter group of the HHV-7 probe is FAM; the fluorescent reporter group of the HPV-11 probe is ROX; the fluorescent reporter group of HPV-16 is CY5; the fluorescence reporter group of the internal standard probe is HEX.
Further, the 3' end of the probe also has a non-fluorescent quencher.
Further, the 3' end of the probe also has a quencher group, such as SQ1, BQ1, SQ2 or BQ2.
In a specific embodiment, the 3' terminus of the probe is SQ1.
Further, the dosage of the primer in the composition is 0.05-5 mu M; the dosage of the probe in the composition is 0.05-5 mu M.
In a particular embodiment, the ingredients of the composition of the invention are present in separate packages.
In a particular embodiment, the components of the composition of the invention are present in the same package.
Further, the components of the composition of the present invention are present in a mixed form.
In a second aspect, the present invention provides the use of a composition of the invention as described above in the manufacture of a kit for the detection of lichen planus pathogens which are HHV-7, HPV-11, HPV-16.
In a third aspect, the present invention provides a kit for the detection of lichen planus pathogens comprising a composition of the invention as described above.
Further, the kit also comprises a negative quality control product and a positive quality control product.
In a specific embodiment, the negative quality control is DEPC H 2 O, physiological saline and internal standard gene pseudovirus. The positive quality control product is at least one of fragment plasmids, DNA fragments and pseudoviruses of HHV-7, HPV-11 and HPV-16 target genes.
Further, the kit also comprises dNTP (U), PCR buffer solution and Mg 2+ At least one of (1).
Still further, the kit further comprises: at least one of a nucleic acid releasing agent, a nucleic acid extracting agent, and a DNA polymerase.
Further, the kit further comprises a nucleic acid releasing reagent, a nucleic acid extracting reagent, dNTP (U) s, DNA polymerase, PCR buffer solution and Mg 2+ At least one of (1).
Further, the concentration of the DNA polymerase is 3U/reaction to 15U/reaction, for example, the DNA polymerase may be Taq enzyme.
In a particular embodiment, the kit of the invention comprises:
PCR buffer, DNA polymerase, UNG enzyme, dNTP (U) s, primers, probes, and metal cations required for catalyzing the DNA polymerase.
Common PCR buffers are Tris-HCl, mgCl 2 And buffer systems such as KCl and Triton X-100. The total volume of the PCR reaction tubes is generally 20-200. Mu.l.
In a specific embodiment, the kit of the present invention is compatible with a digital PCR amplification system, i.e., can be directly used for amplification on a digital PCR instrument.
In a fourth aspect, there is provided a method for detecting lichen planus pathogens, the method comprising the steps of:
1) Extracting or releasing nucleic acid of a sample to be detected;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;
3) Results were obtained and analyzed.
In the present invention, the sample for detection may be a pharyngeal swab, an oropharyngeal swab, vaginal discharge, blood, or the like, but is not limited thereto.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
UNG enzyme reaction at 50-60 deg.c for 1-5 min for 1 circulation; activating Taq enzyme at 90-95 ℃ for 1-5 minutes for 1 cycle; denaturation at 90-95 deg.C for 5-20 s, annealing at 55-60 deg.C for 10-60 s, and collecting fluorescence after 30-50 cycles.
In a particular embodiment, there is provided a method for detecting lichen planus pathogens for non-diagnostic purposes, the method comprising the steps of:
1) Extracting or releasing nucleic acid of a sample to be detected;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;
3) Results were obtained and analyzed.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
UNG enzyme reaction is carried out for 1 to 5 minutes at the temperature of between 50 and 60 ℃ for 1 cycle; activating Taq enzyme at 90-95 ℃ for 1-5 minutes for 1 cycle; denaturation at 90-95 deg.C for 5-20 s, annealing at 55-60 deg.C for 10-60 s, and collecting fluorescence after 30-50 cycles.
As used herein, the term "non-diagnostic purpose" refers to information that is not intended to indicate whether an individual is infected with HHV-7, HPV-11 and HPV-16 and suffers from lichen planus. For example, the method can be used to detect the presence of the pathogen in a test culture in an experiment designed for scientific research.
Drawings
FIG. 1 is a graph showing the results of detection of the composition of the present invention (HHV-7 detection contains only SEQ ID NOS: 1 to 3);
FIG. 2 is a graph showing the results of detection of the composition of the present invention (HHV-7 detection contains SEQ ID NOS: 1-3 and 31-33);
FIG. 3 is a graph showing the results of detection of comparative example composition 1 of the present invention;
FIG. 4 is a graph showing the results of detection of comparative example composition 2 of the present invention.
Detailed Description
The present invention will be specifically explained below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are illustrative of the invention and are not to be construed as limiting the invention.
Example 1 primers and probes used in the present invention
TABLE 1
Wherein, the fluorescence reporter group of the HHV-7 probe is FAM; the fluorescent reporter group of the HPV-11 probe is ROX; the fluorescent reporter group of HPV-16 is CY5; the fluorescent reporter group of the internal standard probe is HEX.
Example 2 method for detecting human lichen planus pathogens
Preparation of reagents:
according to the number of the sample to be detected, the positive control and the negative control, taking the PCR reaction solution and the enzyme mixed solution according to the corresponding amount in proportion (38 mu L/part of the PCR reaction solution and 2 mu L/part of the enzyme mixed solution), fully and uniformly mixing to obtain the PCR mixed solution, and centrifuging at 2000rpm for 10s for later use.
Sample handling and application
200. Mu.L of the sample to be tested, the negative control and the positive control were put into a 1.5mL centrifuge tube, and nucleic acid extraction was carried out using nucleic acid extraction or purification reagents (S10015) from Santa Clarit Biotechnology Ltd, according to the procedures described therein.
The processed sample, negative control and positive control are respectively added into corresponding 0.2mL PCR reaction tubes by sucking 10 uL of each sample, 40 uL of PCR mixed solution is added into each tube, and the tube cover is covered.
And (3) PCR reaction system:
TABLE 2
PCR amplification
PCR amplification is carried out on PCR instruments such as ABI7500 fluorescent quantitative PCR instrument, life Technologies Quant student 5 fluorescent PCR instrument, SLAN-96P full-automatic medical PCR analysis system and the like according to a certain temperature and time setting program. The preferred embodiment of the present invention is shown in Table 3.
TABLE 3
* Note: for ABI7500 instrument reasons, it cannot be set for 30 seconds, and can be set for 31 seconds or 32 seconds.
Interpretation of test results
If the FAM, HEX (VIC), ROX and CY5 channels of the sample have obvious S-type amplification curves and the Ct value is less than or equal to 40, the sample is judged to be positive; if the FAM, ROX and CY5 channels of the sample have No amplification curve (No Ct) or Ct value more than 40, and the HEX (VIC) internal standard channel is positive (the Ct value is less than or equal to 40), the sample is judged to be negative. Specifically, as shown in table 4 below.
TABLE 4
Example 3 test results of test specimens of the composition of the invention
The primer and probe shown in example 1 were used to verify pseudovirus samples by the method of example 2, and the results showed that HHV-7, HPV-11 and HPV-16 could be distinguished, as shown in FIG. 1 (containing only SEQ ID NOS: 1 to 3 for detecting HHV-7) and FIG. 2 (containing SEQ ID NOS: 1 to 3 and 31 to 33 for detecting HHV-7).
Example 4 sensitivity of the compositions of the invention
HHV-7, HPV-11 and HPV-16 virus gene plasmids are diluted to 1.00E +04 copies/mL, 1.00E +03 copies/mL, 4.00E +02 copies/mL, 2.00E +02 copies/mL by using negative skin exudate swab samples in a gradient way to be used as samples to be detected, the detection level of 95 percent is used as the lowest detection limit of the kit, and the analysis sensitivity test result shows that the detection limit of the kit on the HHV-7 virus is as follows: 2.00E +02 copies/mL (see Table 5); the detection limit for HPV-11 virus is: 1.00E +03 copies/mL (see Table 6); the detection limit for HPV-16 virus is: 1.00E +03 copies/mL (see Table 7).
TABLE 5 HHV-7 reagent limits of detection determination test results TABLE-skin exudate sample
TABLE 6 determination of HPV-11 reagent detection limits test results TABLE-skin exudate samples
TABLE 7 determination of detection limits of HPV-16 reagents Table skin exudate sample
Example 5 specificity of the compositions of the invention
The primers and probes shown in example 1 were tested for common infectious viruses (hepatitis B virus (HBV), hepatitis C Virus (HCV), human Immunodeficiency Virus (HIV), treponema Pallidum (TP), herpes simplex virus pathogen type 1, type 2 (HSV-1, HSV-2, HHV-6, etc.), human papilloma virus type 6, type 26, type 42, type 44, type 61 (HPV-6, 26, 42, 44, 61), gonococcus, candida albicans, etc. according to the method of example 2, and the results are shown in Table 8, indicating no cross-reaction to the above pathogens.
TABLE 8
Example 6 interference resistance and stability of the compositions of the invention
In order to examine the influence of possible endogenous/exogenous substances in a sample on a detection result, diluting a pathogen DNA quantitative reference substance with a set value to the lowest detection limit, dividing the pathogen DNA quantitative reference substance into a plurality of parts, and adding endogenous interfering substances with certain concentrations into each part: hemoglobin, white blood cells, cervical mucus, total bilirubin, triglycerides, total IgG, interferon alpha; exogenous interfering substances: ribavirin, cimetidine, ganciclovir, cidofovir, famciclovir, foscarnet, acyclovir, marimbavir, CMV423, contraceptive gel, vaginal lavage fluid, 2% clotrimazole antifungal cream, 4% miconazole antifungal cream and foscarnet, and the anti-interference capability of the kit is examined by comparing with a control sample. The test results are shown in table 9. As can be seen from the table, the samples containing various interfering substances are all positive through detection, and the Ct value is not obviously different from the control, so that the detection result of the kit is not obviously interfered by the interfering substances possibly existing in the samples under the experimental condition of the kit.
TABLE 9
Comparative example 1 primers and probes of the present invention
In the base complementary pairing principle, a dimer is formed between the primer and (or) the probe, but this probability is small and can be excluded at the beginning of the design. However, when multiple pathogens are jointly detected, a large number of primers and probes exist, dimers are easy to occur among the primers, the probes and the probes or between the primers and the probes, the design conservation is ensured (the conservation is crucial to the detection accuracy), the mutual interference among different primers and probes is considered, and the primers and the probes need to be designed and verified carefully.
In addition, the inventor also designs other primers and probes (the sequences are shown in tables 10-11) to form different detection systems 1 and 2 which are also used for triple joint detection of HHV-7, HPV-11 and HPV-16. The specific detection results are shown in fig. 3-4, and it can be seen from the figures that only a part of amplification curves appear in the detection, the amplification curves have low amplification and poor repeatability, and other targets even have no amplification curves, so that the overall detection effect is poor.
Watch 10
TABLE 11
Claims (10)
1. A composition for detecting a predisposition to develop lichen planus, comprising:
upstream and downstream primers and probes for detecting HHV-7 as shown in SEQ ID NO 1-3;
the upstream and downstream primers and probes for detecting HPV-11 as shown in SEQ ID NO 4-6; and
the upstream and downstream primers and probes for detecting HPV-16 are shown in SEQ ID NO 7-9.
2. The composition as claimed in claim 1, further comprising: the upstream and downstream primers and probes for detecting HHV-7 shown in SEQ ID NO: 31-33.
3. The composition as claimed in claim 1, further comprising: and detecting upstream and downstream primers and probes of the internal standard.
4. The composition of claim 3, wherein the upstream and downstream primers and probes of the internal standard are an internal standard upstream primer shown in SEQ ID NO. 10, an internal standard downstream primer shown in SEQ ID NO. 11, and an internal standard probe shown in SEQ ID NO. 12.
5. The composition of claim 1, wherein the fluorophores of the probes of the composition are different from each other and do not interfere with each other.
6. Composition according to any one of claims 1 to 5, characterized in that the components of the composition are present in a mixed form.
7. Use of a composition according to any one of claims 1 to 6 in the manufacture of a kit for the detection of lichen planus pathogens which are HHV-7, HPV-11, HPV-16.
8. A kit for detecting lichen planus pathogens comprising the composition of any one of claims 1-6.
9. The kit of claim 8, further comprising a nucleic acid releasing reagent, a nucleic acid extracting reagent, dntps, a DNA polymerase, a PCR buffer, and Mg 2+ At least one of (1).
10. A method for detecting lichen planus pathogens for non-diagnostic purposes, the method comprising the steps of:
1) Extracting or releasing nucleic acid of a sample to be detected;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition according to any one of claims 1 to 6 or the kit according to claim 8 or 9;
3) Results were obtained and analyzed.
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| US20200385821A1 (en) * | 2019-06-07 | 2020-12-10 | Chapter Diagnostics, Inc. | Methods and compositions for human papillomaviruses and sexually transmitted infections detection, identification and quantification |
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