Primer group for simultaneously detecting four types of horse digestive tract bacteria and application of primer group

By designing a multiplex PCR primer set and a real-time quantitative PCR platform, the problems of low efficiency and poor specificity in the detection of bacteria in the equine digestive tract were solved, enabling rapid, simple, and low-cost detection of four types of bacteria, suitable for horse farms and veterinary hospitals.

CN121975962APending Publication Date: 2026-05-05NANJING ZHUOYI BIOTECHNOLOGY CO LTD
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2026-02-24
Publication Date
2026-05-05

AI Technical Summary

Technical Problem

Existing methods for detecting bacteria in the equine digestive tract are inefficient, have poor specificity, and are costly, making it difficult to quickly and comprehensively screen for four common pathogens, especially due to the lack of specific primer designs for Lawsonia intracellularis.

Method used

A set of multiplex PCR primers was designed to target specific genes of Salmonella enteritidis, Salmonella typhi, Clostridium difficile, and Lawsonia intracellularis. Combined with a real-time quantitative PCR platform, multiplex PCR reaction and singlex PCR confirmation were used to achieve rapid and convenient detection of the four bacteria.

Benefits of technology

This method enables the simultaneous detection of four equine digestive tract bacteria in a single PCR reaction. It is highly sensitive, low-cost, suitable for large-scale sample screening, reduces operation time and reagent costs, and provides accurate and reliable test results.

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Abstract

The invention discloses a primer group for simultaneously detecting four equine digestive tract bacteria and application thereof, the primer group comprises primer sequences as shown in SEQ ID NO: 1-8, and the four equine digestive tract bacteria are salmonella enteritidis, salmonella typhimurium, clostridium difficile and lawsonia intracellular. Extracting total DNA (deoxyribonucleic acid) of a sample to be detected by using the excrement sample nucleic acid extraction kit; the total DNA is used as a template, and the primer group is used for multiple PCR reaction to obtain an amplification curve. According to the invention, a primer sequence with high sensitivity and specificity is adopted, so that the quality of a detection result is ensured; the detection method is simple to operate, time-saving and labor-saving; the detection flux is high, and the reagent consumable cost is low.
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Claims

1. A primer set for simultaneous detection of four types of equine digestive tract bacteria, characterized in that, The primer set includes primer sequences shown in SEQ.ID.NO.1-8, and the four equine digestive tract bacteria are Salmonella enteritidis, Salmonella typhi, Clostridium difficile, and Lawsonia intracellularis.

2. Use of the primer set according to claim 1 in the preparation of a reagent for simultaneously detecting four equine digestive tract bacteria.

3. A kit for simultaneously detecting four types of equine digestive tract bacteria, characterized in that, The kit contains the primer set shown in SEQ.ID.NO.1-8.

4. The kit for simultaneous detection of four equine digestive tract bacteria according to claim 3, characterized in that, It also includes a nucleic acid extraction reagent for the test sample and 2×Universal SYBR qPCR Mix.

5. A method for detecting equine digestive tract bacteria for non-diagnostic purposes, characterized in that, The method includes the following steps: (1) Extract the DNA of the test sample; (2) Using the sample DNA as a template and using the primer set according to claim 1 or the kit according to claim 3, perform a multiplex PCR reaction to obtain an amplification curve after multiplex PCR amplification.

6. The method for detecting equine digestive tract bacteria for non-diagnostic purposes according to claim 5, characterized in that, The reaction system includes 10 μL of 2×Universal SYBR qPCR Mix, 2 μL of the mixed solution of the upper and lower primers of the four digestive tract bacteria, and 8 μL of the DNA template of the test sample to be detected.

7. The method for detecting equine digestive tract bacteria for non-diagnostic purposes according to claim 5, characterized in that, The reaction amplification conditions are pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing / extension at 60°C for 30 s, and 45 cycles of reaction.

8. The method for detecting equine digestive tract bacteria for non-diagnostic purposes according to claim 5, characterized in that, Obtain the amplification curve after multiplex PCR amplification, and judge whether there are digestive tract bacteria according to the Ct value of the amplification curve: If the Ct value of the amplification curve of the test sample > 38 or NoCt, the interpretation result is negative, indicating that there are no above-mentioned digestive tract bacteria in the test sample; If the Ct value of the amplification curve of the test sample is: 0 < Ct < 38, the interpretation result is positive, indicating that the test sample contains one or more of the above-mentioned digestive tract bacteria.