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Multiple fluorescent PCR method and kit for simultaneous detection of human adenovirus, human mycoplasma pneumonia and bocavirus

A human boca virus and human adenovirus technology, applied in the field of biotechnology applications, can solve the problems of difficulty in detecting trace nucleic acids and accurate quantification, cumbersome operations, and long time consumption.

Active Publication Date: 2014-08-20
BEIJING CENT FOR DISEASE PREVENTION & CONTROL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the classic method of detecting corresponding pathogens at home and abroad is the isolation and cultivation of viruses, but its operation is cumbersome and time-consuming; electron microscopy, immunohistochemistry, ELISA, conventional PCR, etc. have their own outstanding advantages, but it is difficult to detect trace amounts of nucleic acid and Accurate quantification, the real-time fluorescent quantitative PCR technology (Real-time fluorescent quantitative PCR) developed at the end of the 20th century is a method of adding fluorescent groups to the PCR reaction system and using the accumulation of fluorescent signals to monitor the entire PCR process in real time. Qualitative and quantitative templates, and has the characteristics of high sensitivity, stronger specificity and reliability, ability to experiment with multiple reactions, high degree of automation, no pollution, real-time and accuracy, etc.

Method used

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  • Multiple fluorescent PCR method and kit for simultaneous detection of human adenovirus, human mycoplasma pneumonia and bocavirus
  • Multiple fluorescent PCR method and kit for simultaneous detection of human adenovirus, human mycoplasma pneumonia and bocavirus
  • Multiple fluorescent PCR method and kit for simultaneous detection of human adenovirus, human mycoplasma pneumonia and bocavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Design and synthesis of specific primers and probes for human adenovirus representative strain Hexon gene, human mycoplasma pneumoniae representative strain RP gene, and human bocavirus representative strain NP1 gene

[0036] 1.1 Selection of Hexon gene of representative human adenovirus strain, RP gene of representative human Mycoplasma pneumoniae strain, NP1 gene of representative human Boca virus strain and determination of conserved regions

[0037] The sequences used in this study were selected from NCBI GenBank (http: / / www.ncbi.nlm.nih.gov), and the main selection criteria were: (a) strains with a relatively recent age; (b) strains with full-length sequences; ( c) Select a representative strain after comparing the sequence with the subtype; (d) Select a strain that infects humans preferentially. The information of the Hexon gene of the representative human adenovirus strain, the RP gene of the representative human Mycoplasma pneumoniae strain, and the N...

Embodiment 2

[0052] Example 2. The establishment of a one-step multiplex fluorescent PCR detection system for human adenovirus, human mycoplasma pneumoniae, and human bocavirus

[0053] 2.1 According to Invitrogen's nucleic acid extraction kit ( DNA Mini Kit) to extract sample nucleic acid.

[0054] 2.2 Preparation of human adenovirus, human mycoplasma pneumoniae, and human bocavirus one-step multiplex fluorescent PCR detection system The preparation reagent used AgPath-IDTM One-step PCR Kit from Ambion Company, and the system was 50 μl:

[0055] 2×PCR buffer 25μl

[0056] 25×PCR Enzyme 2μl

[0057] Detection Enhancer 3μl

[0058] Add 0.5 μl of primers shown in SEQ NO: 1, 2, 4, 5, 7, 8, 10, and 11 at the same time for primers and probes, and the final concentration of primers is 300 nM; add primers such as SEQ NO: 3, 6, 9, 0.5 μl each of the probe primers indicated in 12, the final concentration is 150 nM

[0059] Template 6 μl (add 2 μl for each of the three positive templates, and ...

Embodiment 3

[0067] Example 3. Specific identification of human adenovirus, human mycoplasma pneumoniae, and human bocavirus one-step multiple fluorescent PCR detection method

[0068] The one-step multiplex fluorescent PCR reaction system established in Example 2 is used for human adenovirus, human mycoplasma pneumoniae, human boca virus, influenza A virus, influenza B virus, influenza C virus, respiratory syncytial virus, intestinal The results showed that only human adenovirus, human mycoplasma pneumoniae, and human bocavirus had corresponding specific fluorescence amplification curves in the FAM, JOE, and ROX detection channels, and there was no cross-reaction. In addition to the amplification curve of the internal quality control CY5 channel of the other 5 strains of viruses, the other three channels have no amplification curve, indicating that the method has strong specificity (see Figure 1).

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Abstract

The invention belongs to the field of biological technical application, and relates to a multiple fluorescent PCR method and a kit for simultaneous detection of human adenovirus, human mycoplasma pneumonia and bocavirus and containing internal quality control. The invention designs specific primers and probes aiming at conserved sequences of human adenovirus HP (hexonprotein) gene, human mycoplasma pneumonia RP gene (repetitive element RepMP5e) and bocavirus NP1 (Nucleoprotein) gene, and establishes a one-step multiple fluorescent RT-PCR rapid detection method containing internal quality control; the method has simple and quick operations, overcomes the complexity of single hole single detection in a conventional single fluorescent RT-PCR method, simplifies the operation process, saves test cost, and provides a powerful technical support for on-site virus detection, health assessment and clinical diagnosis by using high specificity, sensitivity, efficiency and stability.

Description

technical field [0001] The invention relates to the application field of biotechnology, in particular, it can be used for the simultaneous detection and identification of human adenovirus, human mycoplasma pneumoniae and human bocavirus. Background technique [0002] Human adenovirus (Human Adenovirus, HAdV) is a group of non-enveloped double-stranded DNA viruses, divided into 1 to 55 serotypes, respectively classified into six serological groups A to F. Human adenovirus spreads through the respiratory tract and digestive tract, causing a variety of infections and causing serious harm to children and immunocompromised persons. The possible diseases caused by adenovirus infection include: acute adenovirus pneumonia, acute gastroenteritis, keratitis, celiac disease, and acute interstitial nephritis. [0003] The human adenovirus genome is about 36kb long, and its capsid (Capsid) has a regular 20-hedron structure with a diameter of about 80-110nm and no envelope. The adenovir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/70C12Q2537/143C12Q2545/101C12Q2563/107
Inventor 崔淑娟田丽丽石伟先张代涛王海滨张玉松李爱华孙玉兰
Owner BEIJING CENT FOR DISEASE PREVENTION & CONTROL
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