Multiplex RT-PCR Combined Gene Chip Detection Kit for Diarrhea Related Pathogens

Abstract

The present invention provides a detection kit for diarrhea-related 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 26 diarrhea-related 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 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 ...

Examples

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 ...

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 ...

Examples

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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