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Diarrhea-related pathogen detection kit combining multiple RT-PCR with gene chip

A detection kit, RT-PCR technology, applied in microorganism-based methods, microbial determination/inspection, resistance to vector-borne diseases, etc., can solve non-specific cross-reaction, high mutation pathogen detection coverage, low detection index less problems

Active Publication Date: 2017-08-25
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 detection coverage of highly mutated pathogens by conventional primers and probes by using multiple specific conservative degenerate primer combinations and probe combinations, and solve the parallel detection of more than 20 kinds of diarrhea-related pathogens in a single reaction system At the same time, it solves the problem of non-specific cross-reaction between multiple primers and probes, overcomes the shortcomings of the prior art single-reaction system with fewer detection indicators, time-consuming and labor-intensive defects, and provides a simple, fast, sensitive, and large detection throughput. Diarrhea-associated pathogen gene detection kit

Method used

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  • Diarrhea-related pathogen detection kit combining multiple RT-PCR with gene chip
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  • Diarrhea-related pathogen detection kit combining multiple RT-PCR with gene chip

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Embodiment 1

[0166]Example 1: Multiplex RT-PCR Combined Gene Chip Detection Kit for Diarrhea-Related Pathogens.

[0167] 1) Design and preparation of multiple specificity conservative degenerate primers and probes:

[0168] 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 alignment analyzes the specificity of the candidate primers to avoid cross-reaction with other nucleic acids in the sample (BLAST+); then evaluate the compatibility of primers between different target genes, and evaluate the content Including Tm value similarity, primer dimer tendency and 3' end hybridization tendency (Python program), finally gener...

Embodiment 2

[0177] Embodiment 2: Detection of positive samples of diarrhea-associated pathogens.

[0178] Use a diarrhea-positive sample with the following pathogens as a stool sample: adenovirus, astrovirus, norovirus type GI, norovirus type GII, rotavirus, sapovirus, salmonella, shigella, Campylobacter, Clostridium difficile, Fusobacterium perfringens, Enterotoxigenic Escherichia coli, Enterohaemorrhagic Escherichia coli, Enteropathogenic Escherichia coli, Enteroinvasive Escherichia coli, Enteroaggregative Escherichia coli, Vibrio cholerae , Vibrio parahaemolyticus, Yersinia enterocolitica, Aeromonas hydrophila, Listeria monocytogenes, Enterobacter sakazakii, Staphylococcus aureus, Cryptosporidium, Giardia enterica and dysentery amoeba, a total of 26 positive samples. At the same time, a positive control (26 pathogenic standard nucleic acid molecules and 1 GAPDH endogenous control standard nucleic acid molecule) and a negative control (sterile water) were set up, and the detection was ...

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Abstract

The invention relates to a diarrhea-related pathogen detection kit combining multiple RT-PCR with a gene chip. According to the kit, a multiple-specificity conserved degenerate primer composition and a probe composition are adopted to detect one or more of 26 diarrhea-related pathogens, and at the same time, endogenous control, positive control and negative control are arranged. By the adoption of the diarrhea-related pathogen detection kit combining the multiple RT-PCR with the gene chip, the coverage rate of target sequences of high-mutation pathogens is increased, the problem of non-specificity cross reaction between multiple primers and probes is avoided, the purpose can be achieved that a single reaction system synchronously detects more than 20 kinds of diarrhea-related pathogens, and a detection tool which is simple, sensitive, rapid, large in throughput and capable of multi-index parallel detection is provided for diarrhea-related pathogen detection.

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 diarrhea-related pathogens. Background technique [0002] Diarrhea caused by pathogen infection is a common clinical disease. There are about 2 billion diarrhea cases in the world every year, of which 2.2 million people die, including 1.8 million children. It is an important cause of infant mortality. Diarrhea can occur all year round, especially in summer and autumn. The infection is mostly caused by food or water pollution through fecal-oral transmission. Public health and socioeconomic development. Common infectious pathogens of diarrheal diseases include viruses, bacteria, parasites, etc. The etiology of the diseases and epidemics caused by them is complex. Therefore, rapid, accurate and multi-index pathogen detection will provide sufficient basis for the diagnosis and treatment of diseases, and provide a ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12Q1/14C12Q1/10C12Q1/04C12R1/93
CPCC12Q1/689C12Q1/701Y02A50/30
Inventor 王勇强刘洋张鑫磊
Owner SUZHOU GENEWORKS TECH CO LTD
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