Unlock instant, AI-driven research and patent intelligence for your innovation.

PCR detection system for simultaneously detecting multiple food-borne pathogenic bacteria and construction method

A construction method and pathogenic bacteria technology, applied in the field of biomedical detection, can solve the problems of cumbersome and time-consuming detection methods, low accuracy and sensitivity

Pending Publication Date: 2021-04-23
XUZHOU MEDICAL UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The primary purpose of the present invention is to provide a PCR detection system for simultaneous detection of multiple food-borne pathogenic bacteria, aiming to solve the problems of cumbersome and time-consuming detection methods of existing food-borne pathogenic bacteria, low accuracy and sensitivity;

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • PCR detection system for simultaneously detecting multiple food-borne pathogenic bacteria and construction method
  • PCR detection system for simultaneously detecting multiple food-borne pathogenic bacteria and construction method
  • PCR detection system for simultaneously detecting multiple food-borne pathogenic bacteria and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] The extraction of embodiment 1DNA

[0074] (1) Log in to GenBank and query the target genes of five foodborne pathogens including Staphylococcus aureus, Listeria monocytogenes, Shigella flexneri, Yersinia enterocolitica, and Clostridium difficile in the nucleic acid database the conserved sequence;

[0075] (2) Target the nuc gene of Staphylococcus aureus, the hlyA gene of Listeria monocytogenes, the ipaH gene of Shigella flexneri, the lysP gene of Yersinia enterocolitica and the tpi gene of Clostridium difficile , carrying out the design of specific oligonucleotide primers;

[0076] (3) Submit the designed candidate sequences to GenBank for BLAST comparison, screen out sequences with better specificity, and synthesize oligonucleotide primers respectively;

[0077] (4) Cultivate the standard and clinical sample bacteria of five kinds of bacteria, use the TIANGEN Bacteria Genomic DNA Kit to extract the DNA of all pathogenic bacteria, and use the DNA-containing supernat...

Embodiment 2

[0079] The establishment and optimization of embodiment 2 PCR reaction system

[0080] The embodiment of the present invention carries out following optimization process on the basis of initial detection system, the preparation of the PCR reaction solution of this initial detection system is:

[0081]

[0082] The PCR reaction conditions are as follows:

[0083] Pre-denaturation: 94°C, 5min,

[0084] Denaturation: 94°C, 30s,

[0085] Annealing: T°C, 30s,

[0086] Extension: 72°C, 30s,

[0087] Number of cycles: 30,

[0088] Final extension: 72°C, 7min,

[0089] The temperature T of each bacteria corresponding to the above-mentioned annealing is shown in Table 1 below:

[0090] Table 1 Annealing temperature

[0091]

[0092] The optimization process includes the following steps:

[0093] (1) Single-plex PCR optimization detection system for five bacteria

[0094] Apply the designed and synthesized primers to the above-mentioned initial detection system, and carry...

Embodiment 3

[0154] Sensitivity and specificity detection of embodiment 3 multiplex PCR

[0155] To verify the specificity of the primers, each primer was reacted with five bacterial DNA mixtures. Likewise, DNA from each pathogen was subjected to PCR reactions with a mixture of five primers. The specificity of PCR primers is shown in Table 3. When the primers are specific and the amplified product does not produce non-target bands, it proves that the specificity of PCR amplification is better.

[0156] Table 3 PCR primer specificity

[0157]

[0158] Measure the DNA concentration of all strains using a UV spectrophotometer. DNA was extracted from five bacterial cultures, and each bacterial DNA was diluted 10-fold, then mixed, and subjected to multiplex PCR for sensitivity detection. The detection limits of each genomic DNA in the multiplex PCR were: 228pg for Staphylococcus aureus, 365pg for Listeria monocytogenes, 1.9pg for Shigella flexneri, 223pg for Clostridium difficile, and 22...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a PCR detection system for simultaneously detecting multiple food-borne pathogenic bacteria and a construction method. Primers and annealing temperatures of an initial detection system are optimized through single PCR of five bacteria; the annealing temperature is further optimized to be 50-59.2 DEG C through triple PCR; the annealing temperatures are optimized to be 58 DEG C and 53.8 DEG C through quadruple PCR, the annealing temperature is optimized to be 54 DEG C through quintuple PCR within the range, and the concentration values of the primers used by all the bacteria are further optimized. Finally, a PCR detection system capable of simultaneously detecting multiple food-borne pathogenic bacteria is obtained, the detection system can be used for carrying out parallel detection on multiple target genes, the detection cost is low, and the detection efficiency is greatly improved, wherein the detection limit of staphylococcus aureus is 228pg, the detection limit of listeria monocytogenes is 365pg, the detection limit of shigella flexneri is 1.9 pg, the detection limit of clostridium difficile is 223pg, and the detection limit of yersinia enterocolitica is 37.9 pg.

Description

technical field [0001] The invention belongs to the field of biomedical detection, in particular to a PCR detection system and a construction method for simultaneously detecting multiple food-borne pathogenic bacteria. Background technique [0002] Foodborne diseases seriously endanger public health and cause severe economic losses in many countries and regions. Therefore, it is particularly important to establish simple, rapid and sensitive detection methods. Important food-borne pathogens (such as Staphylococcus aureus, Listeria monocytogenes, Shigella flexneri, Yersinia enterocolitica, Clostridium difficile, etc.) have a high infection rate and rapid transmission. Among them, Shigella spp. is an important pathogen of human intestinal infectious diseases, and affects intestinal homeostasis. The bacillary dysentery (dysentery) caused by Shigella infection has an annual incidence of 164.7 million worldwide. ; Staphylococcus aureus is an important pathogen that causes food p...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/14C12Q1/10C12Q1/06C12N15/11C12R1/445C12R1/145C12R1/01
CPCC12Q1/689C12Q1/686C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 陈莹王永王鑫汪子轩史巧珍
Owner XUZHOU MEDICAL UNIV