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CN-121992111-A - Primer group for simultaneously detecting 4 horse digestive tract parasites and application thereof

CN121992111ACN 121992111 ACN121992111 ACN 121992111ACN-121992111-A

Abstract

The invention discloses a primer group for simultaneously detecting 4 horse digestive tract parasites and application thereof, wherein the primer group comprises primer sequences shown as SEQ.ID.NO. 1-8, and the 4 horse digestive tract parasites are auxiliary roundworms, strongyloides parvulus, giardia lamblia and eimeria. 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
20260508
Application Date
20260224

Claims (8)

  1. 1. The primer group for simultaneously detecting 4 horse digestive tract parasites is characterized by comprising primer sequences shown as SEQ.ID.NO. 1-8, wherein the 4 horse digestive tract parasites are horse paraascariasis, strongyloides columniforms, giardia lamblia and eimeria.
  2. 2. Use of the primer set of claim 1 for preparing a reagent for simultaneous detection of 4 equine digestive tract parasites.
  3. 3. A kit for simultaneously detecting 4 horse digestive tract parasites is characterized by comprising primer groups shown as SEQ ID No. 1-8.
  4. 4. The kit for simultaneous detection of 4-equine digestive tract parasites of claim 3 further comprising a nucleic acid extraction reagent of the sample to be tested, 2 x Universal SYBR qPCR Mix.
  5. 5. A method for detecting parasites of the digestive tract of horses for non-diagnostic purposes, characterized in that it comprises the steps of: (1) Extracting total DNA of a sample to be detected; (2) Using the total 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 a parasite in an equine digestive tract for non-diagnostic purposes according to claim 5 wherein the reaction system comprises 2X Universal SYBR qPCR Mix. Mu.L, 2. Mu.L of a mixed solution of upper and lower primers for 4 parasites in the digestive tract and 8. Mu.L of a DNA template of the sample to be detected.
  7. 7. The method for detecting equine digestive tract parasites for non-diagnostic purposes according to claim 5, wherein the reaction amplification conditions are 95 ℃ pre-denatured for 5min, 95 ℃ denatured for 30s, 60 ℃ annealed/extended for 30s, 45 cycles of reaction.
  8. 8. The method according to claim 5, wherein an amplification curve after multiplex PCR amplification is obtained, and it is determined whether the digestive tract parasite is contained or not 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 36 or NoCt, the interpretation result is negative, and the fact that the sample to be detected is free of the digestive tract parasites is indicated; If the Ct value of the amplification curve of the sample to be detected is 0< Ct <36, the interpretation result is positive, and the sample to be detected is indicated to contain the 1 or more than 1 kind of digestive tract parasites.

Description

Primer group for simultaneously detecting 4 horse digestive tract parasites and application thereof Technical Field The invention relates to the technical field of biology, in particular to a primer group for simultaneously detecting 4 horse digestive tract parasites and application thereof. Background The parasite infection of the digestive tract of the horses is a common health problem in the horse cultivation process, and seriously threatens the growth and development, the production performance and the life safety of the horses. The 4 parasites are common pathogenic bacteria in the digestive tract of horses, wherein the auxiliary roundworms of the horses are biggest intestinal nematodes in the bodies of equine animals, mainly infect foals, can cause emaciation, anemia, diarrhea and abdominal pain of foals, cause intestinal obstruction and even death when serious, the microcinesis is used as the most common parasitic nematode group in the intestinal tracts of the horses, the imago and the larvae act together to cause intestinal mucosa injury and inflammatory reaction, and are represented by chronic diarrhea and weight reduction, thus influencing the athletic ability and the cultivation benefit of the horses, the giardia lamblia is transmitted through polluted drinking water and feed, and is parasitic on the surface of the intestinal mucosa of the horses, thus causing dyspepsia and watery diarrhea, especially causing damage to young horses and horses with low immunity, and the eimeria coccidiosis mainly parasitic on intestinal epithelial cells of the horses, causes coccidiosis, diarrhea and dehydration and has higher death rate. The existing detection method of the equine digestive tract parasites mainly comprises a traditional microscopic examination method, an immunological detection method and a conventional PCR detection method. The microscopic examination method is simple to operate but extremely low in sensitivity, is easy to leak detection on samples with small number of eggs and atypical morphology, cannot accurately distinguish the types of the closely related parasites, is greatly influenced by experience of operators, is difficult to realize simultaneous detection of various parasites due to the fact that the detection specificity is influenced by sample quality, antibody level and reagent quality in immunological detection methods such as ELISA (enzyme-linked immunosorbent assay) although the sensitivity is higher than that of microscopic examination, and is not suitable for rapid screening of large-scale samples, and the conventional PCR detection method is mostly used for single detection, needs to amplify each parasite independently, is complex in operation and low in detection efficiency, cannot be used for quantitative analysis. The existing multiplex PCR technology is applied to pet parasite detection, but the genome characteristics of the parasite in the digestive tract of the horse, the type and content of PCR inhibitor in the sample matrix (feces) are different from those of the pet, and the detection sensitivity is reduced and the false positive rate is increased by directly applying the existing primer set and detection system. Therefore, development of a multiple detection technology with high specificity, high sensitivity and simple operation for 4 common parasites in the digestive tract of horses is needed, the problems of insufficient coverage, low efficiency and poor accuracy of the existing detection method are solved, and technical support is provided for accurate prevention and control of the digestive tract parasitic diseases of horses. Disclosure of Invention The invention aims to solve the problems of difficult comprehensive screening, low detection efficiency, insufficient accuracy and the like in the existing detection technology of the parasites in the digestive tract of the horses, and provides a novel primer group and a novel method for detecting the parasites in the digestive tract of the horses, so as to overcome the defects of low detection efficiency, high cost and poor 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 parasite genes, wherein the 4 horse digestive tract parasites comprise horse paraascariasis (PARASCARIS EQUORUM), strongylosis minor (Cyathostomum), giardia lamblia (Giardia lamblia) and eimeria tenella (Eimeria leuckarti), the primer group is designed for specific target genes of each parasite and comprises primer combinations for detecting the ITS-2 ribosomal RNA genes of the horse paraascariasis, ITS-2 ribosomal RNA genes of the strongylosis minor, SSU ribosomal RNA genes of the giardia lamblia and 18S ribosomal RNA gene sequence conservation regions of the eimeria tenella, and particularly comprises primer sequences shown as SEQ ID.NO. 1-8. The invention also provides application of the primer group for detecting