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Multiplex-PCR kit for rapid diagnosis of viral encephalitis

A technology for rapid diagnosis of viral encephalitis, applied in recombinant DNA technology, microbial measurement/testing, DNA/RNA fragments, etc., can solve problems such as complicated operation process, delayed treatment of diseases, false positives and false negatives, and achieve Improve detection efficiency, reduce detection cost, and achieve high sensitivity

Active Publication Date: 2017-06-20
上海捷诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the traditional laboratory detection of VE clinically in my country includes EEG, imaging, virus culture, enzyme-linked immunosorbent assay and other methods. The operation process of such methods is complicated and time-consuming, making it difficult to make a diagnosis in the early stage of the disease and delay the treatment. At the same time, it has the disadvantages of poor specificity, low sensitivity, high false positive and false negative

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  • Multiplex-PCR kit for rapid diagnosis of viral encephalitis
  • Multiplex-PCR kit for rapid diagnosis of viral encephalitis
  • Multiplex-PCR kit for rapid diagnosis of viral encephalitis

Examples

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Embodiment 1

[0033] Example 1 Specificity and Sensitivity Detection of Multiplex System

[0034] 1. Specific detection of multiplex system

[0035] Multiplex PCR kit for detection of viral encephalitis, including conventional multiplex PCR components, and specific primers for common meningitis viruses, the sequence is shown in Table 1, conventional multiplex PCR components include Taq polymerase, dNTP, MgCl 2 , PCR buffer, deionized water.

[0036] Table 1 Specific primers for viral meningitis

[0037]

[0038] Specific detection of multiplex system: the specificity of each primer is detected by the method of multi-template single primer. PCR reaction: The reaction system is 25 μl, including 12.5 μl of DreamTaq Green PCR Master Mix (2×), 2 μl of primers (the final concentration of each primer is 0.8 μM / μl), 1.5 μl of mixed genome template (from the prior art, The concentration of each template is 10^4 copies), and the deionized water is made up to a total volume of 25 μl. Cycle condit...

Embodiment 2

[0043] Example 2 Detection of clinical specimens

[0044] From 2014 to 2015, 15 patients suspected of VE but not clearly diagnosed were collected from the First Affiliated Hospital of Soochow University, including 9 males and 6 females, aged 15-60 years old, all of whom had varying degrees of fever, lethargy, and vomiting , diarrhea, listlessness, disturbance of consciousness and other symptoms. Genomic DNA extraction kit (QIAamp DNA Mini Kit) was used to extract DNA from CSF of 15 patients as PCR template. PCR reaction: The reaction system is 25 μl, including 12.5 μl of DreamTaq Green PCRMaster Mix (2×), 3 μl of mixed primers (a total of 12 primers, the final concentration of each primer for HSVI, CMV, EV71, VZV, and EBV is 0.5 μM / μl, The final concentration of each HSVⅡ primer is 0.8 μM / μl), 2 μl template, and deionized water to make up to a total volume of 25 μl. Cycle conditions: preheating at 95°C for 4min, denaturation at 95°C for 30s, annealing at 60°C for 30s, extens...

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Abstract

The invention discloses a multiplex-PCR kit for rapid diagnosis of viral encephalitis. With the application of the multiplex-PCR kit, six viral encephalitis, namely herpes simplex virus type 1 (HSVI), herpes simplex virus type 2 (HSVII), varicella-zoster virus (VZV), EB virus (EBV), enterovirus type 71 (EV71) and cytomegalovirus (CMV), can be rapidly detected; the HSVI, the HSVII, the EBV, the CMV, the EV71 and the VZV are specifically amplified by virtue of six pairs of primers provided by the invention, the primers are free from mutual interference, and the sensitivity of a constructed mPCR system reaches 102-103 copies; for 15 cases of cerebrospinal fluid specimens from patients with suspected clinical viral encephalitis, 6 parts of positive specimens, including 5 cases of HSVI and 1 case of CMV, are detected by virtue of the mPCR system; the mPCR system designed by the invention has the advantages of being high in sensitivity, specificity and stability and being simple to operate; and the six common viruses can be simultaneously detected rapidly; therefore, the multiplex-PCR kit is conducive to rapid diagnosis of the viral encephalitis.

Description

technical field [0001] The invention belongs to the field of gene detection and specifically designs a multiplex PCR kit for rapid diagnosis of viral encephalitis, which can detect six common viral encephalitis pathogens. [0002] technical background [0003] Multiplex PCR is a PCR technique that uses multiple pairs of primers to simultaneously amplify multiple target sequences in the same reaction system on the basis of ordinary PCR. Since multiplex PCR was reported, due to its high efficiency, sensitivity, and economic simplicity, it has been widely used in many fields of nucleic acid diagnosis, such as genetic disease diagnosis, mutation and polymorphism analysis, transgene identification, pathogen detection, etc.; multiplex PCR System, generally includes the following reagents: PCR buffer, Taq enzyme, dNTP, MgCl 2 , primers, template and deionized water. [0004] Viral encephalitis (VE) refers to a central nervous system disease in which one or several viruses infect t...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/701C12Q1/705C12Q2600/16C12Q2537/143C12Q2565/125Y02A50/30
Inventor 孙万平王晓囡季建刚奚邦生刘婉婉张丽珺
Owner 上海捷诺生物科技有限公司
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