CN117305521A - Kit for detecting herpes simplex virus in sample based on MIRA and application thereof - Google Patents
Kit for detecting herpes simplex virus in sample based on MIRA and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological detection, and relates to a kit for detecting herpes simplex virus in a sample based on MIRA and application thereof. The kit comprises a primer pair and a probe for MIRA amplification, wherein the primer pair consists of a forward primer and a reverse primer, and the sequence of the forward primer is as follows: TCACCGGGACGACCACGAGACCGACATGGAGCT, the sequence of the reverse primer is: GCGACCCCTCCCGGTAGCCGTAAAACGGGG ACA; the sequence of the probe is as follows: TGGCACACCACCGAC CTCAAGTACAACCCCTCGCGGGTGGAGGCGTTCCA. The kit for detecting the herpes simplex virus in the sample based on the MIRA and the application thereof can be used for detecting the herpes simplex virus in the sample with simple operation, strong specificity and high sensitivity.
Description
Technical Field
The invention belongs to the technical field of biological detection, and relates to a kit for detecting herpes simplex virus in a sample based on MIRA and application thereof.
Background
Herpes simplex virus (Herpes Simplex Virus, HSV) infection causes fluid-filled blisters in the skin, lips, genitals, etc., and severe infections of the neonate can cause keratitis or encephalitis, which can lead to persistent neurological dysfunction or death. The herpes simplex virus infection can last for life, and relapse is caused after external stimulation, so that the herpes simplex virus infection not only brings long-term mental and physical trouble to a patient, but also greatly increases medical burden. HSV is classified into 2 subtypes, namely HSV-1 and HSV-2, wherein HSV-1 mainly causes skin mucosa infection outside genitalia, and HSV-2 mainly causes genital tract infection, but HSV-1 has been frequently detected in genital infection in recent years.
Early discovery, early diagnosis, early treatment are important actions to be effective against further HSV infection. Currently, the HSV detection method mainly comprises a direct smear method, a virus separation culture method, a serological detection method, a PCR detection method and the like.
The direct smear method has simple operation and low cost, is easily influenced by subjective factors, often has false positive or false negative, and has higher requirements on the technology and experience of detection personnel.
The virus isolation culture method is a gold standard for identifying HSV infection, but the method is limited by conditions and technical level, and is time-consuming, labor-consuming, high in cost, easy to pollute and the like, and is difficult to meet clinical detection requirements.
In the serological detection method, an enzyme-linked immunosorbent assay (ELISA) is common clinically, and the detection of HSV antigen is very practical for a patient with typical clinical manifestation, but the generation of antibodies needs a window period, for example, igM antibodies can be detected only 10 days after infection, so that the diagnosis has hysteresis, and the diagnosis result can not accurately reflect whether the infection is present. In addition, false positives are also easily generated by sampling manual operations and the like.
PCR detection has been developed at present for multiplex PCR, nested PCR, fluorescent quantitative PCR methods, etc. The sensitivity and specificity of the PCR method are significantly higher than those of the cell culture method, but the operation is complex and time-consuming, requires special instruments and equipment, and requires experienced technicians, making it unsuitable for widespread use in resource-limited environments or field testing.
Therefore, there is a need for a herpes simplex virus detection method that is simple to operate, highly specific, and highly sensitive.
The Multi-enzyme isothermal rapid amplification (Multi-enzyme Isothermal Rapid Amplification, MIRA) is a constant temperature nucleic acid rapid amplification technology based on normal temperature, the technology has low requirements on samples and laboratory environments, a small amount of nucleic acid can be amplified to a detectable level in a short time, and a lateral flow chromatography test strip (Lateral flow dipstick, LFD) is combined to form a MIRA-LFD rapid diagnosis method, so that visual reading of results can be realized. The MIRA-LFD method has the advantages of simple and quick operation, high sensitivity, high specificity, no need of special instruments and the like, is suitable for quick detection and is suitable for clinical sample detection.
Disclosure of Invention
The invention aims at providing a kit for detecting herpes simplex virus in a sample based on MIRA, which is simple to operate, high in specificity and high in sensitivity.
To achieve this object, in a basic embodiment, the present invention provides a kit for detecting herpes simplex virus in a sample based on MIRA, said kit comprising a primer pair and a probe for MIRA amplification,
the primer pair consists of a forward primer and a reverse primer, wherein the sequence of the forward primer is as follows: TCACCGGGACGACCACGAGACCGACATGGAGCT, the sequence of the reverse primer is: GCGACCCCTCCCGGTAGCCGTAAAACGGGGACA;
the sequence of the probe is as follows:
TGGCACACCACCGACCTCAAGTACAACCCCTCGCGGGTGGAGGCGTTCCA。
the related principle of the invention is as follows:
2006. niall Armes (ASM Scientific Ltd by Cambridge, wellcome Trust Sanger Institute, UK) was the first to put forward the recombinase polymerase amplification technique RPA. The principle of the method is as follows: the protein-DNA complex formed by combining the recombinase and the primer can search homologous sequences in double-stranded DNA, once the primer locates the homologous sequences, chain exchange reaction can occur to form and start DNA synthesis, and exponential amplification is carried out on a target region on a template; the replaced DNA strand binds to a single-stranded binding protein (SSB) preventing further replacement. In this system, a synthesis event is initiated by two opposing primers, and the entire process proceeds very rapidly, typically within 10 minutes, to obtain detectable levels of amplification product.
The method relies mainly on three proteins: recombinant enzymes capable of binding single stranded nucleic acids (oligonucleotide primers), single stranded DNA binding proteins and strand displacement DNA polymerases. The mixture of these three proteins is also active at normal temperature, with an optimal reaction temperature of around 37 ℃.
In summary, the method achieves separation of DNA double strands not by heating but by one enzyme, and the method can produce billions of DNA copies at a constant temperature in a short time.
At present, on the basis of RPA amplification, researchers have developed various amplification detection technologies based on fluorescence RPA, RPA-combined lateral flow chromatography test paper (RPA-LFD) and the like. The RPA-LFD method is that after RPA amplification is finished, the amplified product is added into a side flow chromatography test strip containing colloidal gold, and the result can be observed with naked eyes within a few minutes. A set of multi-enzyme reaction systems similar to RPA, called MIRA-LFD, was also developed by Fangfang Anpu future Biotechnology Inc.
MIRA is a recombinase polymerase amplification technique developed on the basis of RPA. MIRA electrophoresis, MIRA combined immunofluorescence real-time monitoring and MIRA combined lateral flow chromatography test strip amplification technologies have been developed on the basis of MIRA, and the MIRA combined lateral flow chromatography test strip amplification technologies are all based on a multi-enzyme isothermal amplification method, so that template exponential amplification is realized, but the primer and probe designs and the result presentation forms are different.
The common MIRA only needs to use proper forward and reverse primers; and fluorescent real-time monitoring and lateral flow chromatography require designing unique primers and probes, and the application of the probes greatly reduces nonspecific signals in amplification and improves the specificity and sensitivity of the measurement.
Ordinary MIRA can be observed by direct electrophoresis in a manner similar to PCR product detection; the fluorescent real-time monitoring method judges the result through an amplification curve after amplification by a fluorescent quantitative PCR instrument; and the lateral flow chromatography is visually judged by a colloidal gold detection reagent card. Ease of operation is thus: side flow chromatography > fluorescence real-time monitoring method > electrophoresis method; detection time: electrophoresis method > fluorescence real-time monitoring method > lateral flow chromatography method; detection cost: electrophoresis method > lateral flow chromatography method > fluorescence real-time monitoring method.
MIRA-LFD amplification detection systems comprise labeled primers and probes, polymerase, etc., in principle (see FIG. 1, and see: 1, qinzheng Z, ya L, zheng W, et al Rapid On-Site Detection of the Bursaphelenchus xylophilus Using Recombinase Polymerase Amplification Combined With Lateral Flow Dipstick That Eliminates Interference From Primer-Dependent Artifacts [ J ]. Frontiers in Plant Science,2022,13.2, li J, macdonald J. Advances in isothermal amplification: novel strategies inspired by biological processes [ J ]. Biosensors and Bioelectronics,2015,64 ]):
(1) MIRA reaction: a special primer for RPA-LFD reaction is designed, and the 5' -end of the reverse primer is provided with a biotin label. The probe carries a FAM tag at the 5 'end and a C3-spacer at the 3' end, with a Tetrahydrofuran (THF) spacer in between for endonuclease (nfo) recognition. The recombinant enzyme and the primer form a protein/single-stranded nucleotide complex Rec/ss-DNA, a complementary sequence is scanned and searched on target nucleic acid, a strand displacement reaction can be started after the complementary sequence is found, and a double-stranded DNA template is invaded with the help of auxiliary protein and single-stranded binding protein; forming a D-loop region at the invasion site and starting to scan the DNA double strand; after finding the target region complementary to the primer, the complex Rec/ssDNA disintegrates and the polymerase is bound to the 3' end of the primer to start chain extension, and the target nucleic acid is amplified within 20min by adding a specific molecular probe designed according to the template under the action of nfo. After the reaction, amplified products with FAM and biotin labels are produced.
(2) Detection of lateral flow chromatography test paper: the amplified product is added into the sample well, the FAM-labeled amplified product binds to FAM antibody in the binding region, is driven by capillary force to move through the conjugate pad and bind to anti-FAM-AuNP, and when the amplified product continues to flow to the detection line (T line), the biotin-labeled amplified product is captured by streptavidin in the T line, and a large amount of colloidal gold is aggregated after the two are combined to form a red band.
In a preferred embodiment, the invention provides a kit for detecting herpes simplex virus in a sample based on MIRA, wherein the reverse primer is a 5' biotin-labeled reverse primer.
In a preferred embodiment, the invention provides a kit for detecting herpes simplex virus in a sample based on MIRA, wherein the probe is labelled with FAM at the 5 'end, spC3 at the 3' end and base C between 33 and 35nt is replaced with THF.
In a preferred embodiment, the invention provides a kit for detecting herpes simplex virus in a sample based on MIRA, wherein the MIRA amplification reaction system formed by the kit comprises the following components: 29.4 Mu.l A Buffer, 2. Mu.l 10. Mu.M forward primer, 2. Mu.l 10. Mu.M reverse primer, 0.6. Mu.l 10. Mu.M probe, 12.5. Mu.l ddH 2 O, 1. Mu.l of DNA template, 2.5. Mu.l of B Buffer.
In a preferred embodiment, the invention provides a kit for detecting herpes simplex virus in a sample based on MIRA, wherein the herpes simplex virus is HSV-1 and/or HSV-2.
In a preferred embodiment, the invention provides a kit for detecting herpes simplex virus in a sample based on MIRA, wherein the kit further comprises a nucleic acid detection test strip matched with the detection of the amplification product of MIRA.
In a preferred embodiment, the invention provides a kit for detecting herpes simplex virus in a sample based on MIRA, wherein the nucleic acid detection test strip is a lateral flow chromatography test strip.
In a preferred embodiment, the invention provides a kit for detecting herpes simplex virus in a sample based on MIRA, wherein the lateral flow chromatographic strip comprises FAM antibodies.
In a preferred embodiment, the invention provides a kit for detecting herpes simplex virus in a sample based on MIRA, wherein the lateral flow chromatography test strip is a colloid Jin Celiu chromatography test strip.
The second object of the invention is to provide the use of the kit for detecting whether the herpes simplex virus exists in a sample, so that the detection of the herpes simplex virus in the sample can be performed with simple operation, strong specificity and high sensitivity.
To achieve this object, in a basic embodiment, the invention provides the use of a kit as described above for detecting the presence or absence of herpes simplex virus in a sample, said use being for non-diagnostic purposes.
The kit for detecting the herpes simplex virus in the sample based on the MIRA and the application thereof have the advantages that the kit is simple to operate, high in specificity and high in sensitivity, and the detection of the herpes simplex virus in the sample can be performed.
The kit can detect 2 subtype HSVs simultaneously in a short time conveniently and quickly, and does not need expensive instruments and equipment; the detection sensitivity is high, and 100 genome copies/μl can be detected at the lowest; the detection specificity is good, and the detection has no cross reaction with HSV homonymous virus.
The kit has higher reference value for diagnosis of herpes simplex virus infection and clinical medication guidance, and is suitable for popularization and application.
Drawings
FIG. 1 is a schematic diagram of a MIRA-LFD amplification detection system.
FIG. 2 is a graph showing the results of the amplification reaction temperature-dependent assay of example 2, in which NTC is a negative control (No Template Control) without template.
FIG. 3 is a graph showing the results of the amplification reaction time-dependent assay in example 2.
FIG. 4 is a graph showing the results of the specificity test in example 3.
FIG. 5 is a graph showing the results of the sensitivity test in example 4.
FIG. 6 is a graph showing the result of agarose gel electrophoresis detection of a clinical sample suspected of being infected with HSV in example 5 after PCR amplification.
FIG. 7 is a graph showing the results of the MIRA-LFD test of the present invention on a clinical sample suspected of being infected with HSV in example 5.
Detailed Description
For a better understanding of the technical solutions and advantages of the present invention, the present invention is further described below by way of examples with reference to the accompanying drawings.
Example 1: primer and probe design
Referring to the gB gene nucleotide sequences of HSV-1 and HSV-2 in GenBank (HSV-1 GenBank accession numbers: U49121.1, FJ593289.1, M14164.1, E00357.1, E03092.1, E03113.1, JQ320080; HSV-2 GenBank accession numbers: HM011303.1, M15118.1, M14923.1), a primer pair and a probe capable of simultaneously detecting HSV-1 and HSV-2 are designed according to the MIRA primer design principle.
The full length of the forward primer and the reverse primer in the primer pair is 33nt, and the 5' -end of the reverse primer is labeled with biotin.
Forward primer: TCACCGGGACGACCACGAGACCGACATGGAGCT;
reverse primer: biotin-GCGACCCCTCCCGGTAGCCGTAAAACGGGGACA.
The probe was labeled at the 5 'end with FAM, at the 3' end with SpC3, and base C between 33 and 35nt was replaced with THF.
The probe sequence is as follows:
FAM-TGGCACACCACCGACCTCAAGTACAACCCCTCG[THF]GGGTGGAGGCGTTCCA-C3 Spacer。
example 2: MIRA-LFD amplification detection condition confirmation
1. Preparation of amplification template
Based on the sequence of HSV-1 standard strain F in NCBI (Genbank accession number: GU 734771.1), a 259bp fragment in the maintainer gene gB was taken and a 259bp gB group was synthesized by the division of biological engineering (Shanghai) Co., ltdAfter plasmid concentration (ng/. Mu.l) was determined for the pUC19-HSV-gB plasmid of the fragment, the copy concentration was calculated according to the following formula: copy/μl=6.02×10 23 Xplasmid concentration (ng/. Mu.l). Times.10 -9 /(plasmid base number X660). Diluting the plasmid to 10 6 Copy/. Mu.l was used as template for experiments.
2. Amplification reaction temperature influence test
A MIRA reaction system was established according to the instructions of a DNA isothermal rapid amplification kit (colloidal gold test strip) (purchased from the Biotechnology Co., ltd. In the future of the Weifang Anpu). Each dry powder reaction tube was charged with 29.4. Mu.l of A Buffer, 2. Mu.l of 10. Mu.M forward primer (final concentration 0.4. Mu.M), 2. Mu.l of 10. Mu.M reverse primer (final concentration 0.4. Mu.M), 0.6. Mu.l of 10. Mu.M probe (final concentration 0.12. Mu.M), 12.5. Mu.l of ddH2O, and 1. Mu.l of DNA template. The mixture was transferred to a 0.2 mL reaction tube containing lyophilized enzyme spheres and 2.5 μl of B Buffer was added. To confirm the optimal reaction temperature, the reaction was carried out at 26℃and 30℃and 34℃and 38℃and 42℃and 46℃for 15min, respectively.
After the reaction, the amplified products were diluted 20-fold, respectively, and 10. Mu.l of the amplified products were added to a 1.5. 1.5 mL centrifuge tube containing 190. Mu.l of water, followed by gentle mixing. 50 μl of the diluted product was added to a sample well of a colloidal gold test strip (purchased from the Biotechnology Co., ltd. In the future of the Weifang Anpu) placed horizontally on a table, and the result was observed after 3-5min at room temperature.
And judging the result according to red stripes positioned on the quality control line and the detection line in the test strip. Positive: two red bands appear on the Test strip, one is positioned on a Control Line (C Line) and the other is positioned on a Test Line (T Line); negative: a red strip appears on the quality control line, and the detection line has no strip; invalidation: the quality control line and the detection line are both strip-free.
The test results are shown in fig. 2, and the results show that: all test strip lines C are provided with red strips, which indicates that the experiment is established; the T line can see red strips at 34-46 ℃, which indicates that the amplification reaction is positive in the temperature range; t line has no red band at 26 ℃ and 30 ℃, and the reaction is negative. Therefore, the optimal reaction temperature was confirmed to be 38 ℃.
3. Amplification reaction time Effect assay
The amplification was performed at 38deg.C for 2min, 5min, 10 min, 15min, 20min, 25 min and 30min, respectively, to confirm the optimal reaction time, and the amplification reaction temperature was the same as above.
The test results are shown in fig. 3, and the results indicate that: when the amplification is carried out for 2min, no red positive strip exists, the red positive strip can appear after 5min, the brightness of the red positive strip is highest within 15min-30min, but the brightness of the red positive strip is not obviously improved after the time is prolonged. The reaction product in the reaction system reaches the highest detection amount at 15min. Therefore, the optimal reaction time was confirmed to be 15min.
Example 3: specificity test
HSV belongs to the subfamily of alpha-herpesviridae, and other members of the same family that commonly infect humans are HCMV (beta-herpesviridae) and EBV (gamma-herpesviridae). HCMV and EBV are selected as controls, and the specificity of the kit is evaluated. Culture extract DNA of HCMV and EBV-infected cells was extracted as a control (HCMV standard DNA, concentration 10 5 cobies/. Mu.l; EBV infected cell B958 DNA at a concentration of 0.7. Mu.g/ml); the pUC19-HSV-gB plasmid was used as a positive control (concentration: 10) 5 copies/. Mu.l). As a template, 1. Mu.l of each DNA was used, and an amplification reaction was carried out in accordance with the MIRA-LFD amplification detection conditions (reaction temperature: 38 ℃ C., reaction time: 15 minutes) determined in example 2. After the reaction was completed, 50. Mu.l of each of the reaction products diluted 20-fold was added to the Hybridtect well, and the results were observed and recorded over 3 to 5 minutes, as shown in FIG. 4. The results show that: all test strip lines C are provided with red strips, which indicates that the experiment is established; the positive control HSV C lines and T lines have red stripes, and the result is positive; however, both the EBV and HCMV experimental products had bands on the C line, and the T line was not band, with negative results. Therefore, the kit has good specificity.
Example 4: sensitivity test
To evaluate the sensitivity of MIRA-LFD detection, pUC19-HSV-gB was used as described above with ddH 2 Diluting O to different concentrations 10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 1 copy/. Mu.l, samples of different concentrations were subjected to respective amplification reactions according to the MIRA-LFD amplification assay conditions (reaction temperature 38 ℃ C., reaction time 15 min) determined in example 2. After the reaction was completed, 50. Mu.l of each of the reaction products diluted 20-fold was added to the Hybridtect well, and the results were observed and recorded over 3 to 5 minutes, as shown in FIG. 5. The results show that: as genome copy number decreases, the red band of detection line T becomes lighter, and a minimum of 10 can be detected 2 Copy/. Mu.l. Therefore, the detection limit of the MIRA-LFD method for measuring HSV is 10 2 Copy/. Mu.l.
Example 5: clinical sample detection
10 samples of female vaginal secretion suspected to be infected with HSV clinically are taken, and DNA is extracted and stored for later use. And (3) carrying out PCR test identification on 10 samples, wherein the samples 1-4 contain HSV-1, the samples 5-9 contain HSV-2, and the sample 10 is a negative sample (neither HSV-1 nor HSV-2). The agarose gel electrophoresis detection results of the samples 1-10 after PCR amplification are shown in FIG. 6.
The above 10 samples were subjected to respective amplification reactions under the MIRA-LFD test conditions (reaction temperature: 38 ℃ C., reaction time: 15 min) determined in example 2. After the reaction was completed, 50. Mu.l of each of the reaction products diluted 20-fold was added to the Hybridtect well, and the results were observed and recorded over 3 to 5 minutes, as shown in FIG. 7. The results show that: the kit is used for detecting clinical samples suspected to be infected with HSV, and the detection result is highly consistent with a classical PCR method.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof. The foregoing examples or embodiments are merely illustrative of the invention, which may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The described embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. The scope of the invention should be indicated by the appended claims, and any changes that are equivalent to the intent and scope of the claims are intended to be encompassed within the scope of the invention.
Claims (10)
1. A kit for detecting herpes simplex virus in a sample based on MIRA is characterized in that: the kit comprises a primer pair and a probe for MIRA amplification,
the primer pair consists of a forward primer and a reverse primer, wherein the sequence of the forward primer is as follows: TCACCGGGACGACCACGAGACCGACATGGAGCT, the sequence of the reverse primer is: GCGACCCCTCCCGGTAGCCGTAAAACGGGGACA;
the sequence of the probe is as follows:
TGGCACACCACCGACCTCAAGTACAACCCCTCGCGGGTGGAGGCGTTCCA。
2. the kit of claim 1, wherein: the reverse primer is a reverse primer marked by 5' -end biotin.
3. The kit of claim 1, wherein: the probe was labeled at the 5 'end with FAM, at the 3' end with SpC3, and base C between 33 and 35nt was replaced with THF.
4. The kit according to claim 1, wherein the MIRA amplification reaction system comprising the kit is as follows: 29.4 Mu.l A Buffer, 2. Mu.l 10. Mu.M forward primer, 2. Mu.l 10. Mu.M reverse primer, 0.6. Mu.l 10. Mu.M probe, 12.5. Mu.l ddH 2 O, 1. Mu.l of DNA template, 2.5. Mu.l of B Buffer.
5. The kit of claim 1, wherein: the herpes simplex virus is HSV-1 and/or HSV-2.
6. Kit according to one of claims 1 to 5, characterized in that: the kit also comprises a nucleic acid detection test strip matched with the detection of the MIRA amplification product.
7. The kit of claim 6, wherein: the nucleic acid detection test strip is a lateral flow chromatography test strip.
8. The kit of claim 7, wherein: the lateral flow chromatography test strip comprises FAM antibody.
9. The kit of claim 7, wherein: the lateral flow chromatography test strip is a colloid Jin Celiu chromatography test strip.
10. Use of a kit according to any one of claims 1 to 9 for detecting the presence or absence of herpes simplex virus in a sample, said use being for non-diagnostic purposes.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110904277A (en) * | 2019-12-25 | 2020-03-24 | 圣湘生物科技股份有限公司 | Compositions, kits and methods for HSV-1 and HSV-2 typing detection |
| CN111733284A (en) * | 2020-05-30 | 2020-10-02 | 潍坊安普未来生物科技有限公司 | Primer, probe, reagent and method for quickly detecting feline parvovirus at normal temperature and isothermal temperature |
| CN113355324A (en) * | 2021-07-13 | 2021-09-07 | 国药(武汉)医学实验室有限公司 | Probe and kit for detecting human adenovirus type 7 and using method thereof |
| CN115747379A (en) * | 2022-12-06 | 2023-03-07 | 上海仁度生物科技股份有限公司 | Real-time fluorescent nucleic acid isothermal amplification detection kit for herpes simplex virus types 1 and 2 and special primer and probe thereof |
| CN115927678A (en) * | 2022-08-24 | 2023-04-07 | 重庆医科大学附属第一医院 | LFD-MIRA primer probe combination, kit and detection method for rapidly detecting acinetobacter baumannii on site |
| US20230332216A1 (en) * | 2020-08-25 | 2023-10-19 | Microgen Laboratories | Methods and reagents for rapid detection of pathogens in biological samples |
-
2023
- 2023-11-28 CN CN202311603597.4A patent/CN117305521A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110904277A (en) * | 2019-12-25 | 2020-03-24 | 圣湘生物科技股份有限公司 | Compositions, kits and methods for HSV-1 and HSV-2 typing detection |
| CN111733284A (en) * | 2020-05-30 | 2020-10-02 | 潍坊安普未来生物科技有限公司 | Primer, probe, reagent and method for quickly detecting feline parvovirus at normal temperature and isothermal temperature |
| US20230332216A1 (en) * | 2020-08-25 | 2023-10-19 | Microgen Laboratories | Methods and reagents for rapid detection of pathogens in biological samples |
| CN113355324A (en) * | 2021-07-13 | 2021-09-07 | 国药(武汉)医学实验室有限公司 | Probe and kit for detecting human adenovirus type 7 and using method thereof |
| CN115927678A (en) * | 2022-08-24 | 2023-04-07 | 重庆医科大学附属第一医院 | LFD-MIRA primer probe combination, kit and detection method for rapidly detecting acinetobacter baumannii on site |
| CN115747379A (en) * | 2022-12-06 | 2023-03-07 | 上海仁度生物科技股份有限公司 | Real-time fluorescent nucleic acid isothermal amplification detection kit for herpes simplex virus types 1 and 2 and special primer and probe thereof |
Non-Patent Citations (2)
| Title |
|---|
| HYUN-JIN KIM 等: "A rapid and simple isothermal nucleic acid amplification test for detection of herpes simplex virus types 1 and 2", JOURNAL OF CLINICAL VIROLOGY, vol. 50, 14 October 2010 (2010-10-14), pages 3 * |
| 顾菊萍等: "环介导等温扩增法检测孕妇血清中单纯疱疹病毒Ⅰ型方法的建立", 苏州大学学报(医学版), no. 04, 25 July 2010 (2010-07-25), pages 763 - 766 * |
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