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Herpes simplex virus type 1 and type 2 typing nucleic acid detection kit

A herpes simplex virus and detection kit technology, which is applied in the field of pathogen detection, can solve the problems of inconvenient use and transportation, inability to achieve uniformity, poor detection specificity, etc., so as to shorten the detection time, avoid false positives, and reduce detection costs. Effect

Pending Publication Date: 2020-05-19
AUTOBIO DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the hot-start Taq enzyme amplification system is used for conventional PCR fluorescence quantitative detection DNA amplification system, and the Taq enzyme and reverse transcription Mlv enzyme amplification system are used for RNA amplification. Unite
In terms of detection time, DNA amplification takes about 1.5 hours, and RNA amplification takes about 2 hours, which takes a long time
In addition, the storage condition of the hot-start Taq enzyme amplification reagent must be stored at -20°C, and it must be transported on dry ice or ice packs, which has a short validity period and is inconvenient during use and transportation.
Existing multiplex PCR technology still has disadvantages such as poor detection specificity and low sensitivity

Method used

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  • Herpes simplex virus type 1 and type 2 typing nucleic acid detection kit
  • Herpes simplex virus type 1 and type 2 typing nucleic acid detection kit
  • Herpes simplex virus type 1 and type 2 typing nucleic acid detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] The present embodiment herpes simplex virus type 1 and type 2 typing nucleic acid detection kits are as follows:

[0081] 1. 20 μL PCR reaction solution 1

[0082] The concentration of each component of PCR reaction solution 1 is as follows:

[0083] 0.4 μM HSV-1 forward primer, 0.4 μM HSV-1 reverse primer, 0.3 μM HSV-1 probe;

[0084] 0.4 μM HSV-2 forward primer, 0.4 μM HSV-2 reverse primer, 0.24 μM HSV-2 probe;

[0085] 0.3 μM human β-globin gene DNA forward primer, 0.3 μM human β-globin gene DNA reverse primer, 0.15 μM human β-globin gene DNA probe;

[0086] 0.5 U / μL DNA polymerase, 0.05 U / μL UDG enzyme, 0.2 mM dATP, 0.2 mM dGTP, 0.2 mM dCTP, 0.4 mM dUTP, 0.1% preservative-sodium azide.

[0087] 50mM tris(hydroxymethyl)methylglycine (pH8.3), 100mM potassium acetate;

[0088] 8% DMSO, 0.05% Tween20, 2% glycerin.

[0089] 2. 10 μL PCR reaction solution 2

[0090] The concentration of each component of PCR reaction solution 2 is:

[0091] 3mM Mn(OAc) 2 , 0.1% pr...

Embodiment 2

[0095] This example is a method for detecting herpes simplex virus type 1 / type 2 DNA nucleic acid in unknown samples such as male urethral secretions and female cervical secretions using the kit prepared in Example 1.

[0096] 1. Reagent preparation

[0097] According to the number of samples to be tested, negative control and positive control, take the corresponding amount of PCR reaction solution 1 and PCR reaction solution 2 according to the ratio (20μL / per person PCR reaction solution 1+10μL / person part PCR reaction solution 2), and mix well Make a PCR-MIX mixture, centrifuge briefly and set aside.

[0098] 2. Sample processing

[0099] 1. Put the swab to be tested into 1mL of normal saline, wash it 10 times, and use the liquid after washing;

[0100] 2. Take 600 μL of the liquid sample after rinsing, and use the magnetic bead method nucleic acid extraction reagent of Zhengzhou Antu Bioengineering Co., Ltd. to extract the herpes simplex virus type 1 / type 2 DNA nucleic ac...

Embodiment 3

[0112] Embodiment 3 kit performance measurement of the present invention

[0113] 1. Sensitivity experiment

[0114] Cooperate with the magnetic bead method nucleic acid extraction reagent of Zhengzhou Antu Bioengineering Co., Ltd., and use the kit prepared in Example 1 to detect the reference product of the enterprise, and the detection rate of the reference product with the detection limit is >95%. This kit detects 10 cases of HSV-1 and HSV-2 detection limit enterprise work reference products, Figure 1a Detect HSV-1 type detection limit reference product amplification curve for kit of the present invention; Figure 1b It is the amplification curve of the reference product for the detection limit of HSV-2 type detected by the kit of the present invention.

[0115] Conclusion: According to the detection results, the kit of the present invention cooperates with Zhengzhou Antu Bioengineering Co., Ltd. magnetic bead method nucleic acid extraction reagent to detect 10 cases of H...

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Abstract

The invention provides a herpes simplex virus type 1 and type 2 typing nucleic acid detection kit which includes a PCR reaction solution 1, a PCR reaction solution 2, negative control and positive control. The PCR reaction solution 1 is prepared from a HSV-1 type upstream primer, a downstream primer, a fluorescent probe, a HSV-2 type upstream primer, a downstream primer, a fluorescent probe, DNA polymerase, uracil DNA glycosylase, dNTP, a PCR buffer, a PCR amplification enhancer, an antiseptic agent and water; and the PCR reaction solution 2 is prepared from manganese acetate, an antiseptic agent and water. The herpes simplex virus type 1 and type 2 typing nucleic acid detection kit is convenient to operate, the reagent is stored at 2-8 DEG C, the detection sensitivity is high, the specificity is good, the repeatability is high, and the detection result is fast. The herpes simplex virus type 1 and type 2 typing nucleic acid detection kit can be used to simultaneously perform typing fluorescence PCR qualitative detection on specific DNA nucleic acid fragments of herpes simplex virus type 1 and type 2 in a single tube, a reliable molecular diagnostic basis is provided for diagnosingherpes simplex virus infection.

Description

technical field [0001] The invention relates to the technical field of pathogen detection, in particular to a herpes simplex virus type 1 and type 2 typing nucleic acid detection kit. Background technique [0002] Herpes simplex virus (Herpes simplex virus, HSV) belongs to the double-stranded DNA virus of the subfamily Alphavirinae of the family Herpesviridae, and is divided into type 1 and type 2 according to the antigenicity, namely HSV-1 and HSV-2, two types of nucleotide sequences There is 50% homology. Type 1 is mainly obtained from lip lesions, and mainly infects the skin and mucous membranes of the head (eyes, mouth, lips), upper trunk skin, and central nervous system, causing herpes simplex encephalitis, herpetic keratitis, oral herpes, dermatitis, etc.; HSV-2 can be isolated from genital lesions, mainly causing skin and mucous membrane herpes in genital area and neonatal infection. Herpes simplex virus is widely distributed in the world, infection is very common i...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6848C12N15/11
CPCC12Q1/705C12Q1/6848C12Q2563/107C12Q2537/143C12Q2531/113
Inventor 胡明明张学增王义聪王玮李振红付光宇杨增利苗拥军
Owner AUTOBIO DIAGNOSTICS CO LTD
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