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Method and kit for detecting genotypes of herpes simplex virus

A herpes simplex virus and genotyping technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, material stimulation analysis, etc., can solve problems such as indistinct melting curve characteristics, low market share, and inability to judge results , to achieve stable and reliable results, ensure specificity of detection, easy and fast operation

Inactive Publication Date: 2011-05-25
SHANGHAI XINGYAO MED TECH DEV CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The genotyping method of HSV usually adopts molecular biological methods, such as strand substitution reaction (SDA) and PCR technology. At present, there are commercialized HSV genotyping detection kits abroad for scientific research or clinical diagnosis, including BD in the United States. , Roche and QIAGEN of Germany’s HSV-1 / 2 genotyping detection kits, of which BD’s products use SDA technology, and the market share is not high; while Roche’s and QIAGEN’s products use FRET probe-based fluorescent PCR technology and Melting curve analysis method, each sample needs to add a melting curve analysis step after fluorescent PCR is completed, and genotype HSV according to the Tm value of the amplified product, which requires many steps and takes a long time for detection
In addition, there are literatures (Anderson TP et al, J ClinMicrobiol, 2003, 41(5): 2135-2137) reporting that the use of melting curves for HSV genotyping has the disadvantage that the characteristics of the melting curve are not obvious and the results cannot be judged

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  • Method and kit for detecting genotypes of herpes simplex virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: the preparation of herpes simplex virus genotyping detection kit (PCR-fluorescent probe method)

[0050] (1) Primer probe synthesis

[0051] Primer probes were designed according to the sequence of SEQ ID No.1 in the content of the invention, and a commercial sequence synthesis company (Shanghai Sangon Bioengineering Technology Service Co., Ltd.) was entrusted to synthesize the following sequences:

[0052] The upstream primer is 5'-TCCATgCgCATCATCTACg-3', as shown in SEQ ID No.2.

[0053] The downstream primer is 5'-TgTggCTCgCCATCTTgT-3', as shown in SEQ ID No.3.

[0054] The HSV-1 fluorescent probe is 5'-ACTCCATATTTgTgCTgTgCCgC-3' (5' end labeled FAM, 3' end labeled BHQ-1), as shown in SEQ ID No.4.

[0055] The HSV-2 fluorescent probe is 5'-TggCCACCAgCgCTTCgC-3' (HEX is marked at the 5' end, and BHQ-1 is marked at the 3' end), as shown in SEQ ID No.5.

[0056] The internal reference probe is 5'-TTCAgCgAgCgTgggACATCAgg-3' (5' labeled Cy5, 3' labeled BH...

Embodiment 2

[0077] Example 2: Application of Herpes Simplex Virus Genotyping Detection Kit (PCR-Fluorescent Probe Method) in Skin Oral Herpes HSV Detection

[0078] (1) Sample processing

[0079] Use a cotton swab to pick up herpes exudate from the patient's skin, eyes or oral mucosa, then put the cotton swab into 1ml of normal saline, shake it well, squeeze the cotton swab dry, and centrifuge the rinse solution at 13,000rpm for 6 minutes. Carefully aspirate the supernatant with a pipette (5 μl of residual liquid can be reserved to prevent aspiration of the precipitate). Add 50 μl of nucleic acid extraction solution to the pellet, vortex to mix, keep at 100°C for 10 minutes, centrifuge at 13,000 rpm for 6 minutes, and take the supernatant for fluorescent PCR amplification detection. The negative and positive controls of the kit are processed in the same way as the sample solution.

[0080] (2) Amplification detection

[0081] According to the method in the summary of the invention, eac...

Embodiment 3

[0083] Embodiment 3: the application of herpes simplex virus genotyping detection kit (PCR-fluorescent probe method) in the detection of genital herpes HSV

[0084] (1) Sample processing

[0085] Use a cotton swab to collect the secretions from the urethral lesion of the patient, then put the cotton swab into 1ml of normal saline, shake it well, squeeze the cotton swab dry, take the rinse solution and centrifuge it at 13,000rpm for 6 minutes, and use a pipette Carefully aspirate the supernatant (to prevent aspiration of the precipitate, 5 μl of residual liquid can be reserved). Add 50 μl of nucleic acid extraction solution to the pellet, vortex to mix, keep at 100°C for 10 minutes, centrifuge at 13,000 rpm for 6 minutes, and take the supernatant for fluorescent PCR amplification detection. The negative and positive controls of the kit are processed in the same way as the sample solution.

[0086] (2) Amplification detection

[0087] According to the method in the summary of...

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Abstract

The invention discloses a method and a kit for detecting genotypes of a herpes simplex virus (HSV). Primer probes designed on the basis of HSV DNA polymerase gene are adopted, type 1 and type 2 of the HSV are detected simultaneously in a single tube by TaqMan probe dual-wavelength fluorescent PCR technology, and the existence and the type of the HSV in a sample are judged through fluorescent signals and circulating threshold values of amplification templates with different wavelengths. In the kit, a manually constructed internal reference system is used for avoiding false negative of a detection result. The kit comprises nucleic acid extracting solution, PCR buffer solution, Taq enzyme, an internal reference, negative control and positive control; and two steps of sample treatment and amplification detection are adopted. The kit is easy and convenient to operate and high in sensitivity, can avoid false positive caused by PCR product nucleic acid pollution, can solve the problem of false negative caused by a PCR inhibitor in the sample, and can be widely applied to quick detection of the genotypes of the pathogen clinically.

Description

technical field [0001] The invention belongs to a method and a kit for genotyping herpes simplex virus by utilizing fluorescent PCR technology. Background technique [0002] Herpes simplex virus (Herpes Simplex Viruses, HSV) belongs to the herpes virus family, and the diameter of the virus particle is about 180nm, with double-stranded DNA. 2 types, and the genome sequencing of HSV-1 and HSV-2 has been completed. Clinically, HSV can cause herpes on the face and lips (facial herpes, such as "oral herpes"), or herpes on the genital area (genital herpes), etc., and HSV infections in other parts of the genitals are mostly caused by HSV-1 (accounting for 95% ), and the HSV infection of reproductive organs is mainly caused by HSV-2 type (accounting for 78%), but both types of HSV can infect these two parts, and both can infect newborns. Generally, HSV can infect newborns through the birth canal during delivery, causing neonatal herpes, skin, eye or oral infection, damage to the c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12R1/93
Inventor 吴大治夏懿
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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