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Real-time fluorescence nucleic acid isothermal amplification detection kit for human herpesvirus 1

A herpes simplex virus, constant temperature amplification detection technology, applied in the field of real-time fluorescent nucleic acid constant temperature amplification detection, primers, probes and related kits of herpes simplex virus type 1 (HSV-1), can solve the problem of inability to distinguish HSV infection Varicella-zoster virus infection, unable to distinguish between HSV-1 and HSV-2, unable to achieve rapid and quantitative detection, etc., to achieve high accuracy, accurate detection, and monitoring of the existence of false negatives

Active Publication Date: 2016-06-01
SHANGHAI RENDU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The culture method is very time-consuming, requires high equipment, and can only examine symptomatic patients. In asymptomatic patient samples, the sensitivity is low, less than 50%, which makes most HSV-infected patients directly missed and cannot be distinguished. HSV-1 and HSV-2; cytological examination cannot distinguish HSV infection or varicella-zoster virus infection, and the specificity is poor; enzyme-linked immunoassay (ELISA) needs to coat the antigen overnight, which cannot achieve rapid and quantitative detection results ; Immunofluorescence assay (IFA) has a sensitivity in the range of 35% to 70% when examining samples from asymptomatic patients, and the results are largely dependent on clinical subjective experience
However, the problems faced by the application of SAT technology in the detection of different types of viruses are different, and it is necessary to specifically analyze the characteristics of the virus for special design.
At present, there is no research report on the real-time fluorescent nucleic acid constant temperature amplification detection technology for herpes simplex virus type 1 (HSV-1)

Method used

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  • Real-time fluorescence nucleic acid isothermal amplification detection kit for human herpesvirus 1
  • Real-time fluorescence nucleic acid isothermal amplification detection kit for human herpesvirus 1
  • Real-time fluorescence nucleic acid isothermal amplification detection kit for human herpesvirus 1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Embodiment 1 is used for the design of special primers and probes of real-time fluorescent nucleic acid constant temperature amplification detection herpes simplex virus type 1 (HSV-1)

[0098] The present invention selects highly conserved and highly specific segments in the HSV-1US5 gene as the amplified target sequence region (its nucleotide sequence is shown in SEQIDNo.: 1), according to the principle of primer probe design, using DNAStar, DNAMAN software And artificial design is used for real-time fluorescent nucleic acid constant temperature amplification and detects the special primer and probe sequence of herpes simplex virus type 1 (HSV-1), obtains following specific sequence:

[0099] (1) a capture probe (TCO, TargetCaptureOligo) that can specifically combine with the target nucleic acid (HSV-1RNA) sequence of herpes simplex virus type 1 (HSV-1), the nucleotide sequence of the capture probe is: 5 'TGCACACCGGATGGCCAATCCAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA3' (SE...

Embodiment 2

[0108] Example 2 Preparation of a real-time fluorescent nucleic acid constant temperature amplification detection kit for herpes simplex virus type 1 (HSV-1)

[0109] Using the special primers and probes provided in Example 1, the real-time fluorescent nucleic acid constant temperature amplification detection kit for herpes simplex virus type 1 (HSV-1) of the present invention was obtained. The kit contains components such as capture probe (TCO, TargetCaptureOligo), T7 primer, nT7 primer, HSV-1 detection probe, M-MLV reverse transcriptase and T7 RNA polymerase, among which:

[0110] The nucleotide sequence of the capture probe is SEQ ID NO:2, the sequence of the T7 primer is SEQ ID NO:3, the sequence of the nT7 primer is SEQ ID NO:4, and the nucleotide sequence of the detection probe is SEQ ID NO:5.

[0111] The capture probe is present in the viral nucleic acid extraction solution, the T7 primer, nT7 primer and HSV-1 detection probe are present in the HSV-1 amplification de...

Embodiment 3

[0127] The specific detection of embodiment 3 kits

[0128] The detection kit made by the present invention is similar to herpes simplex virus type 1 in classification, similar in symptoms, and prone to cross-reaction microorganisms for specific detection. Specifically, samples No. 1-9 are varicella-zoster virus (VZV), EB Virus (EBV), cytomegalovirus (CMV), simian B virus (simianherpesBvirus), herpes simplex virus type 2 (HSV-2), human papillomavirus (HPV), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG ), Ureaplasma urealyticum (UU). The composition and specific method of the kit are as follows:

[0129] 1. Preparation of reference substance

[0130] 1.1 Internal standard: take 400 μL of diluent, add 10 μL of HIV-1 internal standard (10 5 HSV-1ICRNA dilution), mix well and set aside.

[0131] 1.2 Positive control: take 250 μL negative control, add 10 μL positive control substance (10 6 copy / mL dilution of herpes simplex virus type 1 in vitro transcribed RNA), mi...

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Abstract

The invention discloses a real-time fluorescence nucleic acid isothermal amplification detection kit for human herpesvirus 1 (HSV-1). The real-time fluorescence nucleic acid isothermal amplification detection kit specifically comprises a capturing probe, a T7 primer and an nT7 primer of HSV-1, an HSV-1 detection probe, M-MLV reverse transcriptase, T7RNA polymerase, and other reagent. The method provided by the invention can perform nucleic acid isothermal amplification detection on swab, scraping liquid and cerebrospinal liquid samples containing the HSV-1 in a high specificity, high sensitivity, low pollution and rapid manner; and the kit has the characteristics of high detection efficiency and high accuracy, so that the kit has bright application prospects.

Description

technical field [0001] The invention relates to the technical field of biological detection of viruses, in particular to a method used in the real-time fluorescent nucleic acid constant temperature amplification detection of herpes simplex virus type 1 (HSV-1) which combines specific target capture technology and real-time fluorescent nucleic acid constant temperature amplification detection technology Primers, probes and related kits. Background technique [0002] Herpes simplex virus is widely distributed in the world, and the population is extremely susceptible to infection. There are many latent and recurrent infections. Patients and virus carriers are the source of infection of the virus. The exposure route enters the body and resides in the normal mucous membrane, blood, saliva and sensory ganglion cells of the human body. Most of the primary infections are recessive, most of them have no clinical symptoms or show subclinical manifestations, and only a few can appear ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 王瑞靖于明辉居金良
Owner SHANGHAI RENDU BIOTECH
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