Method for detecting 29 respiratory pathogens by using Taqman low-density microfluidic (TAC) chip technology

A microfluidic chip and respiratory technology, applied in the detection of 29 kinds of respiratory pathogens, can solve the problems of unfavorable simultaneous screening of multiple pathogen infections, long bacterial or cell culture cycle, and low sensitivity of ordinary gel electrophoresis

Active Publication Date: 2019-11-05
ACADEMY OF MILITARY MEDICAL SCI
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Problems solved by technology

However, the above-mentioned technologies have more or less certain defects: the bacterial or cell culture cycle is long and the requirements are high, and the diagnosis of pathogens requires a certain timeliness, and the bacterial or cell culture cannot meet the requirements of short-term rapid detection; antigen-antibody reactions The sensitivity and specificity are low, false positives are prone to occur, and it is not suitable for the detection of mixed infections of various pathogens; the sensitivity of ordinary gel electrophoresis is low, sometimes the bands are blurred, the judgment is not accurate enough, and it is easy to cause misdiagnosis
However, there are many commercial kits at home and abroad, and the price is relatively expensive. In addition, they are mainly aimed at the detection of a single respiratory pathogen infection, and there are few methods for simultaneous detection of different respiratory pathogens, which is not conducive to the detection of multiple pathogen infections. Simultaneous screening
In multiplex PCR detection, since the primers and probes for detecting different pathogens are in one reaction tube, it is very easy to cause false positives.
In addition, in the multiplex PCR detection, the change of the primer probe for detecting one of the pathogens will lead to the change of the whole experimental system, which requires re-optimization of the detection system, which makes the experiment more complicated

Method used

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  • Method for detecting 29 respiratory pathogens by using Taqman low-density microfluidic (TAC) chip technology
  • Method for detecting 29 respiratory pathogens by using Taqman low-density microfluidic (TAC) chip technology
  • Method for detecting 29 respiratory pathogens by using Taqman low-density microfluidic (TAC) chip technology

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Embodiment 1, for the primer probe design of 29 kinds of respiratory pathogen detection

[0090] The primer probes for the detection of 29 kinds of respiratory pathogens are shown in Table 1.

[0091] Table 1

[0092]

[0093]

[0094]

[0095] Note: merged base Y stands for C or T; merged base R stands for A or G; merged base S stands for G or C; merged base K stands for G or T. Note: F stands for upstream primer; R stands for downstream primer; P stands for MGB probe. The 5' end of the probe is labeled with the FAM fluorescent group, and the 3' end is labeled with the MGB group. In each set of primer-probe combination, the molar ratio of upstream primer, downstream primer and probe is 1:1:0.5.

[0096] The single-plex real-time quantitative PCR in the present invention uses RNP as an internal reference gene, and TAC detection uses RNP and IPCO as internal reference genes. The primers and probes used to detect IPCO were designed and synthesized by ABI Comp...

Embodiment 2

[0100] Embodiment 2, the preparation of positive plasmid standard

[0101] A positive plasmid standard for each pathogen was prepared for 29 pathogens.

[0102] Insert the target fragment into the cloning vector pEASY-Blunt zero (PEASY-Blunt zero Cloning Kit, full gold, lot number CB501) to obtain a positive plasmid standard (sequencing verification).

[0103] The target fragments corresponding to each pathogen are shown in Table 2.

[0104] Table 2

[0105] pathogen GenBank number of digits INF-A (Influenza A virus) MK995729.1 171-276 INF-B (Influenza B virus) MK999133.1 657-747 PIV-1 (parainfluenza virus type 1) MH684390.1 778-859 PIV-2 (parainfluenza virus type 2) MH892406.1 7463-7619 PIV-3 (parainfluenza virus type 3) KT765995.1 291-549 RSV-A (respiratory syncytial virus type A) MH760599.1 1083-1166 RSV-B (respiratory syncytial virus type B) MK749916.1 1275-1377 HMPV-A (human metapneumovirus type A...

Embodiment 3

[0106] Example 3, Preliminary Evaluation of Single-plex Real-time Quantitative PCR of Primer Probe

[0107] 1. Detection of clinical isolates

[0108]Strains to be tested: 29 pathogenic strains isolated clinically.

[0109] CB (Rickettsia, Coxiella burneti) is recorded in the literature: He Zemin, Sun Zhihui, Yu Yonghui et al. Cloning and identification of phase II mutant of Coxiella burneti strain Jiuli [J]. Biotechnology Communications, v.30 (2):170-174. Publicly available from the Academy of Military Medicine, Academy of Military Science, Chinese People's Liberation Army.

[0110] HEV (Enterovirus, Human Enterovirus) is recorded in the literature: Wo Ying. 2012-2014 Analysis of Epidemiological Characteristics of Respiratory Virus Infection in Children in Chongqing [D]. Academy of Military Medical Sciences of the Chinese People's Liberation Army, 2015. Obtained by the Institute of Medicine.

[0111] MV (Measles Virus, Measles Virus) is recorded in the literature: Zhu Xiao...

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Abstract

The invention discloses a method for detecting 29 respiratory pathogens by using a Taqman low-density microfluidic chip (TAC) technology. According to the method, the TAC detection technology is established for the 29 respiratory pathogens, the advantages of MGB probe detecting are fully used, the defects of previous multiple detection of the respiratory pathogens are overcome, 29-respiratory-pathogen multi-target detection of the respiratory pathogens can be achieved on a sample in two hours, and the good sensitivity and the good specificity are achieved. According to the method for detectingthe 29 respiratory pathogens by using the TAC technology, a quick, accurate and high-throughput technological mean is provided for monitoring and outbreak investigation of respiratory diseases in China.

Description

technical field [0001] The invention relates to a method for detecting 29 kinds of respiratory pathogens by using Taqman low-density microfluidic chip technology. Background technique [0002] Respiratory diseases include acute respiratory infection and community-acquired pneumonia, which pose serious threats and challenges to human health. Among them, respiratory pathogens closely related to the occurrence of the two types of diseases include viruses, bacteria, mycoplasma, chlamydia, and rickettsia, etc. Many pathogens cause similar clinical symptoms, and misdiagnosis is easy to occur in pathogen diagnosis, resulting in the widespread phenomenon of antibiotic abuse exist. Timely and accurate differential diagnosis of pathogens is helpful for the prevention and treatment of respiratory diseases and reduces the social medical and health burden. [0003] For a long time, the traditional respiratory pathogen detection technology mainly includes bacterial and virus culture, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/689C12Q1/6851C12Q1/04C12N15/11
CPCC12Q1/701C12Q1/689C12Q1/6851C12Q2600/16C12Q2565/629C12Q2561/101Y02A50/30
Inventor 刘鹏姜永强刘可可律清宇郑玉玲江华孔德聪
Owner ACADEMY OF MILITARY MEDICAL SCI
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