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CN-121975962-A - Primer group for simultaneously detecting 4 horse digestive tract bacteria and application thereof

CN121975962ACN 121975962 ACN121975962 ACN 121975962ACN-121975962-A

Abstract

The invention discloses a primer group for simultaneously detecting 4 horse digestive tract bacteria and application thereof, wherein the primer group comprises a primer sequence shown as SEQ ID NO. 1-8, and the 4 horse digestive tract bacteria are salmonella enteritidis, salmonella typhi, clostridium difficile and lawsonia intracellularis. Extracting total DNA of the sample to be detected by using a fecal sample nucleic acid extraction kit, and performing multiplex PCR reaction by using the total DNA as a template and using a primer group to obtain an amplification curve. The invention adopts a primer sequence with high sensitivity and specificity to ensure the quality of detection results, and the detection method has the advantages of simple operation, time and labor saving, high detection flux and low reagent consumable cost.

Inventors

  • WANG YANBIN
  • LI YONG

Assignees

  • 南京卓一生物科技有限公司

Dates

Publication Date
20260505
Application Date
20260224

Claims (8)

  1. 1. The primer group for simultaneously detecting 4 horse digestive tract bacteria is characterized by comprising primer sequences shown as SEQ.ID.NO. 1-8, wherein the 4 horse digestive tract bacteria are salmonella enteritidis, salmonella typhi, clostridium difficile and lawsonia intracellularis.
  2. 2. Use of the primer set of claim 1 for preparing a reagent for simultaneous detection of 4 equine digestive tract bacteria.
  3. 3. A kit for simultaneously detecting 4 horse digestive tract bacteria is characterized by comprising primer groups shown as SEQ.ID.NO. 1-8.
  4. 4. The kit for simultaneous detection of 4-horse digestive tract bacteria according to claim 3, further comprising a nucleic acid extraction reagent of a sample to be detected, 2× Universal SYBR qPCR Mix.
  5. 5. A method for detecting bacteria in the digestive tract of horses for non-diagnostic purposes, characterized in that it comprises the steps of: (1) Extracting DNA of a sample to be detected; (2) Using the sample DNA as a template and using the primer set of claim 1 or the kit of claim 3, performing multiplex PCR reaction to obtain an amplification curve after multiplex PCR amplification.
  6. 6. The method for detecting equine digestive tract bacteria for non-diagnostic purposes according to claim 5, wherein the reaction system comprises 2.times. Universal SYBR qPCR Mix. Mu.L, 2.mu.L of the 4 kinds of digestive tract bacteria upper and lower primer mixture solution and 8.mu.L of the sample DNA template to be detected.
  7. 7. The method for detecting equine digestive tract bacteria for non-diagnostic purposes according to claim 5, wherein the reaction amplification conditions are 95 ℃ pre-denaturation for 5min, 95 ℃ denaturation for 30s, 60 ℃ annealing/extension for 30s, 45 cycles of reaction.
  8. 8. The method for detecting equine digestive tract bacteria for non-diagnostic purposes according to claim 5, wherein an amplification curve after multiplex PCR amplification is obtained, and whether or not the digestive tract bacteria are contained is judged based on the Ct value of the amplification curve: If the Ct value of the amplification curve of the sample to be detected is more than 38 or NoCt, judging that the result is negative, and indicating that the sample to be detected is free of the digestive tract bacteria; if the Ct value of the amplification curve of the sample to be detected is 0< Ct <38, the result of the interpretation is positive, and the sample to be detected is indicated to contain the 1 or more than 1 kind of digestive tract bacteria.

Description

Primer group for simultaneously detecting 4 horse digestive tract bacteria and application thereof Technical Field The invention relates to the technical field of biology, in particular to a multiplex qPCR primer group for simultaneously detecting 4-horse digestive tract bacteria and application thereof. Background The bacterial infection of the digestive tract of the horses is one of important diseases which endanger the health of the horses, the 4 bacteria are common pathogenic bacteria of the digestive tract of the horses, and the 4 bacteria can be transmitted through contaminated feed, drinking water or contact to cause serious digestive tract diseases. Salmonella enteritidis can invade intestinal epithelial cells of horses to cause symptoms such as fever, vomiting and diarrhea, dehydration and electrolyte disorder are caused when serious, especially the damage to young horses is remarkable, salmonella typhi has stronger pathogenicity, can be planted in horses for a long time to cause chronic infection, is manifested as emaciation, anemia and intestinal ulcers, and even causes systemic infection through blood transmission, clostridium difficile is used as anaerobic bacillus, can generate various toxins to destroy intestinal mucosa barriers to cause pseudomembranous enteritis, is manifested as severe diarrhea and abdominal pain, has high infection recurrence rate and high treatment difficulty, and lawsonia intracellularis mainly hosts in the intestinal epithelial cells of horses to cause proliferative enteritis, is manifested as chronic diarrhea, weight loss and dyspepsia, and seriously influences the growth performance and cultivation benefits of horses. The existing detection method of the equine digestive tract bacteria mainly comprises a microorganism culture method, an immunological detection method and a conventional PCR detection method. The microorganism culture method is a traditional gold detection standard, but has the advantages of complicated operation, long culture period (usually 3-7 days), difficulty in meeting the requirement of rapid diagnosis, severe culture conditions of partial bacteria, easiness in false negative, cross reaction risk of immunological detection methods such as ELISA, insufficient specificity and limited detection sensitivity for low-concentration bacteria, most of conventional PCR detection methods are single detection, multiple amplification is required to finish detection of various bacteria, operation is complex, efficiency is low, and the method is not suitable for large-scale sample screening due to the subsequent steps such as gel electrophoresis. The existing multiplex PCR technology has been applied to the detection of intestinal bacteria of pets, but the genome characteristics of the digestive tract bacteria of horses and the interference factors in sample matrixes are different from those of pets, and the design of specific primers aiming at horse-specific pathogenic bacteria (such as epidemic strains of lawsonia intracellularis in horses) is still blank. Therefore, development of a high-specificity, high-sensitivity, rapid and convenient multiple detection technology for 4 common pathogenic bacteria in the digestive tract of horses is needed, the problems of low efficiency, poor specificity and high cost of the existing detection method are solved, and technical support is provided for precise prevention and control of digestive tract bacterial diseases of horses. Disclosure of Invention The invention aims to solve the problems of difficult comprehensive screening, low detection efficiency, high detection cost and the like in the existing detection technology of the equine digestive tract bacteria, and provides a novel primer group and a novel method for detecting the equine digestive tract bacteria, so as to overcome the defects of low detection efficiency, high cost and insufficient specificity in the related detection technology. In order to achieve the aim, the invention provides a multiplex PCR primer group for simultaneously detecting 4 horse digestive tract bacteria genes, wherein the 4 horse digestive tract bacteria comprise salmonella enteritidis (Salmonella enteritidis), salmonella typhi (Salmonella enterica serovar Typhimurium), clostridium difficile (Clostridioides difficile) and lawsonia intracellularis (Lawsonia intracellularis), the primer group is designed aiming at specific target genes of each bacteria and comprises primer combinations for detecting salmonella enteritidis invA genes, salmonella typhi STM4497 genes, clostridium difficile tpi genes and lawsonia intracellularis fox gene sequence conservation regions, and the primer combinations specifically comprise primer sequences shown as SEQ ID.NO. 1-8. The invention also provides application of the primer group for detecting the equine digestive tract bacteria by multiplex PCR in preparation of a reagent for detecting the equine digestive tract bacteria. A kit for simultaneously detecti