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Multiplex RT-PCR Combined Gene Chip Detection Kit for Common Respiratory Tract Pathogens

A technology of RT-PCR and detection kits, which is applied in the direction of microorganism-based methods, resistance to vector-borne diseases, and detection/inspection of microorganisms, which can solve the problems of time-consuming, laborious, non-specific cross-reaction, and low detection coverage of highly mutated pathogens And other issues

Active Publication Date: 2020-03-20
SUZHOU GENEWORKS TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to solve the problem of low coverage of conventional primers and probes for the detection of highly mutated pathogens by using multiple specific conservative degenerate primer combinations and probe combinations, and solve the problem of parallel detection of more than 20 respiratory pathogens in a single reaction system problem, solve the problem of non-specific cross-reaction between multiple primers and probes at the same time, overcome the defects of less detection indicators and time-consuming and labor-intensive defects of the single reaction system in the prior art, and provide a respiratory system with simple operation, fast sensitivity, and large detection throughput Pathogen Gene Detection Kit

Method used

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  • Multiplex RT-PCR Combined Gene Chip Detection Kit for Common Respiratory Tract Pathogens
  • Multiplex RT-PCR Combined Gene Chip Detection Kit for Common Respiratory Tract Pathogens
  • Multiplex RT-PCR Combined Gene Chip Detection Kit for Common Respiratory Tract Pathogens

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Embodiment 1

[0146] Example 1: Multiplex RT-PCR Combined Gene Chip Detection Kit for Common Respiratory Tract Pathogens.

[0147] 1) Design and preparation of multiple specificity conservative degenerate primers and probes:

[0148] Download the nucleic acid sequences of each pathogen from the nucleic acid database, perform multiple sequence comparisons (NCBI database, ClustalW), and generate a degenerate sequence (Python program) according to the comparison results; remove the highly variable regions in the sequence (Python program), and pass The primer design process generates multiple pairs of candidate primers (Primer3, Python program), and sequence comparison analyzes the specificity of the candidate primers to avoid cross-reaction with other nucleic acids in the sample (BLAST+); then evaluate the compatibility between the primers of different target genes, and evaluate the content Including Tm value similarity, primer dimer tendency and 3' end hybridization tendency (Python program),...

Embodiment 2

[0157] Embodiment 2: Detection of positive samples of respiratory pathogens.

[0158] Use respiratory positive samples with the following pathogens, the sample type is throat swab sampling or lung lavage fluid: influenza virus type A, influenza virus type B, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3 , parainfluenza virus type 4, human metapneumovirus, rhinovirus type A, rhinovirus type B, rhinovirus type C, bocavirus, adenovirus, coronavirus 229E, coronavirus HKU-1, coronavirus NL63, coronavirus OC43, respiratory syncytial virus type A, respiratory syncytial virus type B, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila and Streptococcus pneumoniae, a total of 22 positive samples. At the same time, a positive control (22 pathogenic standard nucleic acid molecules and 1 GAPDH endogenous control standard nucleic acid molecule) and a negative control (sterile water) were set, and the detection was carried out in the same...

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Abstract

The present invention provides a detection kit for common respiratory tract pathogens combining multiple RT-PCR with a gene chip. The kit uses a multispecific, conservative degenerate primer combination and a probe combination to detect one or more of 22 common respiratory tract pathogens, and is provided with an endogenous control, a positive control, and a negative control.

Description

technical field [0001] The invention relates to the technical field of nucleic acid detection, in particular to a multiple RT-PCR combined gene chip detection kit for common respiratory pathogens. Background technique [0002] Acute respiratory infection is a common clinical disease in my country, which can cause serious complications and even death. Common infectious pathogens include viruses, bacteria, mycoplasma, chlamydia, etc. The etiology of diseases and epidemics caused by them is complex. Therefore, rapid, accurate and multi-index pathogen detection will provide sufficient basis for disease diagnosis and treatment, and provide a basis for epidemic control. [0003] Currently, commonly used respiratory pathogen detection techniques include pathogen antigen detection and nucleic acid detection. Enzyme-linked immunosorbent assay (ELISA) and immunological test strips are the main protein-based detection methods at present. This type of method is convenient and fast to u...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12Q1/14C12Q1/04C12R1/93
CPCC12Q1/689C12Q1/701Y02A50/30
Inventor 王勇强刘洋张鑫磊
Owner SUZHOU GENEWORKS TECH CO LTD
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