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Multiplex PCR detection kit for 12 encephalitis virus nucleic acids and application thereof

A detection kit, a technology for encephalitis virus, which is applied in the direction of determination/inspection of microorganisms, microorganisms, biochemical equipment and methods, etc., can solve problems such as interaction, and achieve the effects of high detection sensitivity, guaranteed reliability, and simple operation.

Active Publication Date: 2016-03-09
NANJING MOKOBIO BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] The technical problem solved by the present invention: In order to solve the problem of interaction between multiple pairs of primers in the same reaction system, the present invention adopts the multiplex PCR amplification technology of general primers

Method used

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  • Multiplex PCR detection kit for 12 encephalitis virus nucleic acids and application thereof
  • Multiplex PCR detection kit for 12 encephalitis virus nucleic acids and application thereof
  • Multiplex PCR detection kit for 12 encephalitis virus nucleic acids and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 112

[0047] Embodiment 112 kinds of encephalitis virus nucleic acid multiplex PCR detection kits

[0048] The kit is composed of PCR reaction buffer, enzyme system, positive control substance, negative control substance and DEPC water. The enzyme system contains reverse transcriptase (200U / ul), RNase inhibitor (80U / ul), A mixture of hot start TaqDNA polymerase (5U / ul); the PCR reaction buffer contains 5 × onestepRT-PCRBuffer (Tris-HClpH8.5100mM, KCl500nM, MgCl 2 15nM), Mg 2+ (25mM), dNTPs (25mM), 12 pairs of chimeric primers and 1 pair of universal primers;; the positive control consists of 10 of 12 targets 7 copy / mLPMD19-T clone DNA composition; negative control is saline.

[0049] The final concentration of the universal primer in the amplification system is 800nM; the final concentration of the chimeric primer in the amplification system is 60nM.

[0050] The reaction system of the kit is 25ul, specifically as follows: 2.0 μL of enzyme system, 18.0 μL of PCR reaction buffer, ...

Embodiment 2

[0051] Operation and result judgment of embodiment 2 kit

[0052] (1) Extraction of viral genomic DNA

[0053] Use Tiangen Biochemical Technology Co., Ltd.’s viral genome DNA / RNA extraction kit (article number: DP312) to extract nucleic acids from cerebrospinal fluid and serum samples in the sample processing area according to the instructions.

[0054] (2) Preparation of reaction system

[0055] The following experiment was carried out using the kit of Example 1. After the PCR reaction solution of the kit was completely dissolved at room temperature, it was shaken and mixed quickly. The 25 μL PCR reaction system was: 18 μL of PCR reaction buffer, 2 μL of enzyme system, template (including sample extraction) Nucleic acid, negative control and positive control) 3 μL, add DEPC water to 25 μL.

[0056] (3) PCR amplification

[0057] Put the PCR tube into the ordinary PCR machine, open the hot cover, and set the reaction program of the PCR machine according to the following req...

Embodiment 3

[0063] Embodiment 3 kit specific experiment

[0064] Select 8 kinds of virus samples of coxsackie virus, poliovirus, human herpesvirus type 7, human parvovirus B19, polyomavirus BK, influenza virus, adenovirus, and echovirus, and use the kit described in Example 1 According to the method described in Example 2 for operation and result determination, the above 8 kinds of virus detection results showed that there were no characteristic peaks with peak sizes shown in Table 2, indicating that the kit of the present invention had good specificity.

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Abstract

The invention discloses a multiplex PCR detection kit for 12 encephalitis virus nucleic acids and application thereof. The multiplex PCR detection kit comprises primers used for amplifying specific gene sites of the following 12 encephalitis virus: JeV, HTLV1, WNV, HSV1, HSVII, HHV6, JCV, HTLV2, BBF, HCMV, EBV and VZV. The detection kit has the advantages of capacity of realizing multiplex detection, high sensitivity and rapidness and convenience in usage. Moreover, specific primer sequences are employed to guarantee reliability of detection results. A detection method is simple to operate and labor-saving and time-saving; detection flux is great, and cost for reagent consumables is low; nucleic acids extracted from encephalitis pathogen samples can be directly detected; and requirements on a detection platform and manual technique levels are low, so the method can be extensively popularized in conventional detection.

Description

technical field [0001] The invention belongs to the field of gene detection, and relates to a kit for detecting 12 kinds of encephalitis virus nucleic acids by using a universal primer-mediated multiplex PCR amplification method and an application thereof. Background technique [0002] Viral encephalitis is an infectious disease of the central nervous system caused by a variety of viruses. It can occur all year round, so it is also called sporadic encephalitis. The incidence rate is about (3.5-7.4) / 100,000. It has been increasing year by year. Viral encephalitis occurs mostly in children. The disease often lacks specific clinical manifestations. In severe cases, the brain parenchyma is severely damaged, which can lead to neurological sequelae and even death. At present, it is believed that the detection of virus, viral nucleic acid or virus-specific antibody in cerebrospinal fluid (Cerebrospinal fluids, CSF) can clarify the etiology of central nervous system virus infection....

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2565/125C12Q2525/10
Inventor 刘文干金鑫朱坤管海华
Owner NANJING MOKOBIO BIOTECH
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