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Method and kit for detecting nucleic acid with herpes simplex virus

A herpes simplex virus and detection method technology, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of detection of herpes simplex virus that have not been seen yet, and achieve reasonable design and guaranteed detection Specificity, reasonable effect of technical solutions

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

At present, there is no kit for rapid detection of herpes simplex virus and internal reference at the same time using single-tube dual-wavelength real-time fluorescent PCR

Method used

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  • Method and kit for detecting nucleic acid with herpes simplex virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: the preparation of herpes simplex virus nucleic acid detection kit (PCR-fluorescent probe method)

[0049] (1) Primer probe synthesis

[0050] 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:

[0051] The upstream primer is 5'-gAgAgggACATCCAggACTTTg-3', as shown in SEQ ID No.2;

[0052] The downstream primer is 5'-TgAgCTTgTAATACACCgTCAgg-3', as shown in SEQ ID No.3;

[0053] The fluorescent probe for herpes simplex virus is 5'-TCACCgCCgAACTgAgCAgACACC-3' (5' labeled FAM, 3' labeled TAMRA), as shown in SEQ ID No.4.

[0054] The internal reference probe is 5'-TTCAgCgAgCgTgggACATCAgg-3' (5' labeled HEX, 3' labeled TAMRA), as shown in SEQ ID No.5.

[0055] Dissolve them in sterile double-distilled water to a concentration of 25 μM, and store at -20°...

Embodiment 2

[0075] Embodiment 2: the application of herpes simplex virus nucleic acid detection kit (PCR-fluorescent probe method)

[0076] (1) Sample processing

[0077] 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.

[0078] (2) Amplification detection

[0079] According to the method in the summary of the invention, each component of the unseal...

Embodiment 3

[0081] Embodiment 3: the application of herpes simplex virus nucleic acid detection kit (PCR-fluorescent probe method)

[0082] (1) Sample processing

[0083] 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.

[0084] (2) Amplification detection

[0085]According to the method in the summary of the invention, each component of the ...

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Abstract

The invention discloses a method and a kit for identifying a herpes simplex virus (HSV) by utilizing fluorescent PCR. Primer probes designed on the basis of HSV DNA polymerase gene and a manually constructed internal reference system are adopted, the herpes simplex virus and internal reference DNA are detected in a single tube by TaqMan probe dual-wavelength fluorescent PCR technology, and the existence of the HSV in a sample is judged through fluorescent signal intensity and a circulating threshold value of an amplification template. 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 pathogen clinically.

Description

technical field [0001] The invention belongs to a method and a kit for detecting herpes simplex virus by fluorescent PCR. 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. 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 central nervous system and other internal organs, resulting in mental retardation, and e...

Claims

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Application Information

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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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