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

Abstract

The invention discloses a primer group for simultaneously detecting four types of horse alimentary canal parasites and application of the primer group. The primer group comprises primer sequences as shown in SEQ.ID.NO.1-8, and the four types of horse alimentary canal parasites are ascaris horse parasite, strongylus arvensis, giardia blue and eimeria. 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.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a primer set for the simultaneous detection of four types of equine digestive tract parasites and its application. Background Technology

[0002] Intestinal parasitic infections in horses are a common health problem in horse farming, seriously threatening their growth, development, production performance, and even their lives. The four parasites mentioned are common pathogens in the equine digestive tract: *Parascaris equina*, the largest intestinal nematode in equines, primarily infects foals, causing emaciation, anemia, diarrhea, and abdominal pain, and in severe cases, intestinal obstruction and even death; *Rhizoctonia solani*, the most common parasitic nematode group in the equine intestine, can cause intestinal mucosal damage and inflammation through the combined action of adults and larvae, manifesting as chronic diarrhea and weight loss, affecting the horse's athletic ability and farming efficiency; *Giardia lamblia*, transmitted through contaminated drinking water and feed, parasitizes the surface of the small intestinal mucosa, causing indigestion and watery diarrhea, especially severe in young horses and those with weakened immune systems; and *Eimeria tenella*, which mainly parasitizes the intestinal epithelial cells of horses, causing coccidiosis, manifesting as hemorrhagic enteritis, diarrhea, dehydration, and a high mortality rate.

[0003] Currently, the main methods for detecting equine intestinal parasites include traditional microscopic examination, immunological detection, and conventional PCR. Microscopic examination involves observing eggs or cysts through direct smears or saturated saline flotation. While simple to operate, it has extremely low sensitivity, easily resulting in missed detections in samples with few eggs or atypical morphology. Furthermore, it cannot accurately distinguish between closely related parasites, and the results are heavily influenced by the operator's experience. Immunological detection methods, such as ELISA, while more sensitive than microscopic examination, carry the risk of cross-reactivity. Sample quality, antibody levels, and reagent quality can all affect the specificity of the detection, and it is difficult to simultaneously detect multiple parasites. Conventional PCR is mostly a single-parasite detection method, requiring separate amplification for each parasite. This is cumbersome, inefficient, and cannot provide quantitative analysis, making it unsuitable for rapid screening of large-scale samples.

[0004] Multiplex PCR technology has been applied in the detection of parasites in pets, but the genomic characteristics of equine digestive tract parasites and the types and amounts of PCR inhibitors in the sample matrix (feces) differ from those in pets. Directly applying existing primer sets and detection systems leads to decreased detection sensitivity and increased false positive rates. Therefore, there is an urgent need to develop a highly specific, highly sensitive, and easy-to-use multiplex detection technology for four common equine digestive tract parasites to address the problems of incomplete coverage, low efficiency, and poor accuracy of existing detection methods, and to provide technical support for the precise prevention and control of equine digestive tract parasitic diseases. Summary of the Invention

[0005] The purpose of this invention is to solve the problems of difficulty in comprehensive screening, low detection efficiency and insufficient accuracy in existing equine digestive tract parasite detection technologies, and to provide a new primer set and method for equine digestive tract parasite detection, so as to improve the defects of low detection efficiency, high cost and poor specificity in related detection technologies.

[0006] To achieve the above objectives, this invention provides a multiplex PCR primer set for the simultaneous detection of genes from four equine gastrointestinal parasites, namely, *Parascaris equorum*, *Cyathostomum*, *Giardia lamblia*, and *Eimeria leuckarti*. The primer set is designed for specific target genes of each parasite, including primer combinations for detecting conserved regions of the *Parascaris equorum* ITS-2 ribosomal RNA gene, *Cyathostomum* ITS-2 ribosomal RNA gene, *Giardia lamblia* SSU ribosomal RNA gene, and *Eimeria leuckarti* 18S ribosomal RNA gene sequences, specifically including primer sequences as shown in SEQ ID NO. 1-8.

[0007] The present invention also provides the use of the above-mentioned primer set for multiplex PCR detection of equine digestive tract parasites in the preparation of reagents for detecting equine digestive tract parasites.

[0008] A kit for simultaneous detection of four equine gastrointestinal parasites, the kit comprising primer sets as shown in SEQ.ID.NO.1~8, and also including nucleic acid extraction reagents for the test samples and 2×Universal SYBR qPCR Mix.

[0009] The method for detecting equine digestive tract parasites provided by this invention includes the following steps:

[0010] (1) Total DNA was extracted from the sample to be tested using a fecal sample nucleic acid extraction kit;

[0011] (2) Using the total DNA as a template, perform multiplex PCR using the primer set or kit described in this invention. Mix the four primers in equal volumes (the initial concentration of a single primer is 100 μol / L) to prepare a primer mixture solution. The reaction system includes 10 μL of 2×Universal SYBR qPCR Mix, 2 μL of the above four digestive tract parasite primer mixture solution, and 8 μL of DNA template from the sample to be tested.

[0012] (3) The reaction amplification conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing / extension (collecting fluorescence signal) for 30 s, for 45 cycles, to obtain the amplification curve after multiplex PCR amplification;

[0013] (4)Judgment of detection results:

[0014] If the Ct value of the amplification curve of the test sample > 36 or NoCt, the test result is judged as negative, indicating that there is no above-mentioned digestive tract parasite in the test sample;

[0015] If the Ct value of the amplification curve of the test sample is 0 < Ct < 36, the test result is judged as positive, indicating that the test sample contains one or more of the above-mentioned digestive tract parasites, and a second single qPCR test is required to determine the specific parasite species;

[0016] (5)Secondary detection: Using the DNA of the test sample with a positive first detection as a template, use the single-parasite primer described in this invention, and perform a single qPCR reaction according to the above reaction system and amplification conditions to obtain an amplification curve:

[0017] If the Ct value of the amplification curve of primer pair SEQ.ID.NO.1 / SEQ.ID.NO.2 is 0 < Ct < 36, it indicates the presence of Parascaris equorum;

[0018] If the Ct value of the amplification curve of primer pair SEQ.ID.NO.3 / SEQ.ID.NO.4 is 0 < Ct < 36, it indicates the presence of Strongylus equinus;

[0019] If the Ct value of the amplification curve of primer pair SEQ.ID.NO.5 / SEQ.ID.NO.6 is 0 < Ct < 36, it indicates the presence of Giardia lamblia;

[0020] If the Ct value of the amplification curve of primer pair SEQ.ID.NO.7 / SEQ.ID.NO.8 is 0 < Ct < 36, it indicates the presence of Eimeria.

[0021] This method uses a fecal sample nucleic acid extraction kit to extract nucleic acids from the sample, which can effectively remove PCR inhibitors in the feces; the provided primer set is specifically designed and optimized, there is no cross-amplification reaction between primers, and no dimers are generated, ensuring the credibility of the detection results.

[0022] Beneficial effects

[0023] This invention provides a primer set for simultaneously detecting 4 kinds of equine digestive tract parasites and a multiplex PCR detection method based on a real-time fluorescence quantitative PCR platform, which has the following advantages:

[0024] (1)Comprehensive multiplex detection coverage: It can simultaneously screen 4 common parasites in the equine digestive tract in one PCR reaction, covering nematodes and protozoa groups, fully meeting the parasite screening needs in horse breeding, quickly obtaining detection results, and saving time, manpower and material costs;

[0025] (2) High detection rate: The primers are specifically designed and optimized, and have high conservation and specificity. The detection rate is significantly higher than that of traditional microscopic examination, and can accurately distinguish the types of parasites, avoiding missed detection and misjudgment.

[0026] (3) High sensitivity: The detection sensitivity of each target parasite can reach 0.0005 ng / ul, which can detect low concentrations of parasite nucleic acid and is suitable for screening early infection;

[0027] (4) Lower cost: Multiplex PCR testing only requires two operations to complete screening and confirmation, saving more than 70% of reagent costs and operation time compared with single-multiplex testing, and reducing labor costs; Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0029] Figure 1 These are the primer-specific detection results of this invention, where 1 is a sample containing bacteria, and 2-6 are Salmonella, Escherichia coli, Streptococcus equi, Pasteurella, and Mycoplasma, respectively.

[0030] Figure 2 These are the primer sensitivity test results of this invention, where 1-5 are mixed templates of different concentrations (5ng / ul, 0.5ng / ul, 0.05ng / ul, 0.005ng / ul, 0.0005ng / ul). Detailed Implementation

[0031] To better understand the present invention, the following embodiments further illustrate its content; however, the scope of protection of the present invention is not limited to the following embodiments. Any non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are also within the scope of protection of the present invention.

[0032] 1. DNA extraction from the sample to be tested

[0033] Horse feces contain adult worms, eggs, or cysts of intestinal parasites. This detection method obtains DNA from horse feces samples. DNA extraction is performed using a commercial fecal sample nucleic acid extraction kit (such as a centrifugal column nucleic acid extraction kit), following the kit's instructions. The extracted DNA sample is then stored in a 1.5 mL centrifuge tube at 4°C until use.

[0034] 2. Primer design and synthesis

[0035] Refer to the conserved regions of the ITS-2 ribosomal RNA gene of Parascaris equorum, the ITS-2 ribosomal RNA gene of Strongyloides stercoralis, the SSU ribosomal RNA gene of Giardia lamblia, and the 18S ribosomal RNA gene sequence of Eimeria spp. published in GenBank. Use Primer Premier 6 software to design their respective specific primers, and preliminarily verify the designed primers through BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and Primer BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ).

[0036] All primers were synthesized by Sangon Biotech Co., Ltd. During the design process, align the target genes of related species of the target parasite, and select the conserved regions for primer design; conduct dimer analysis and amplicon analysis on the 4 primer groups to ensure that there is no cross-amplification between different primer groups and avoid false positive test results. The optimized primer sequences are shown in Table 1 below:

[0037] Table 1 Primers for multiplex PCR of 4 equine gastrointestinal parasites

[0038]

[0039] 3. Multiplex PCR reaction

[0040] Mix 4 primers in equal volume (the initial concentration of each primer is 100 μmol / L) to prepare a primer mixture solution. The reaction system is prepared as follows: 10 μL of 2×Universal SYBR qPCR Mix, 2 μL of the above-mentioned primer mixture solution for 4 gastrointestinal parasites, and 8 μL of the DNA template of the sample to be tested.

[0041] Amplification conditions for multiplex PCR reaction: Pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing / extension (collecting fluorescence signals) at 60°C for 30 s, and perform 45 cycles of reaction to obtain the amplification curve after multiplex PCR amplification.

[0042] 4. Judgment of test results

[0043] (1) Multiplex PCR screening:

[0044] If the Ct value of the amplification curve of the sample to be tested > 36 or NoCt, the test result is judged as negative;

[0045] If the Ct value of the amplification curve of the sample to be tested is 0 < Ct < 36, the test result is judged as positive, and secondary single detection is required.

[0046] (2) Single qPCR confirmation:

[0047] The reaction system included 10 μL of 2×Universal SYBR qPCR Mix, 2 μL of single parasite primer mixture (upstream and downstream primers mixed, final concentration 0.5 μM), and 8 μL of DNA template from the sample to be tested; the amplification conditions were the same as for multiplex PCR.

[0048] The specific type of parasite infection is determined based on the Ct value of the amplification curve of different primer pairs, and the determination criteria are as described in the invention.

[0049] 5. Specific detection

[0050] Five irrelevant pathogens (Salmonella, Escherichia coli, Streptococcus equi, Pasteurella multocida, and Mycoplasma) were selected as simulated interference samples, and the primer set of this invention was used for detection under optimized amplification conditions. The results are attached. Figure 1 As shown, samples containing only the target parasite exhibited a typical S-shaped amplification curve, while other unrelated pathogens and mixed samples did not show amplification curves, indicating that the detection method of the present invention has good specificity.

[0051] 6. Sensitivity Testing

[0052] The templates for the four parasites were adjusted to 50 ng / ul, and serially diluted to 5 ng / ul, 0.5 ng / ul, 0.05 ng / ul, 0.005 ng / ul, and 0.0005 ng / ul, respectively, for detection using the primer set of this invention. The results are attached. Figure 2 As shown, the limit of detection for nucleic acids of the four equine digestive tract parasites was 0.0005 ng / ul, indicating that the method has high sensitivity.

[0053] This invention provides a complete solution for the rapid detection of four common equine digestive tract parasites, enabling rapid and accurate detection of infection sources. It saves valuable time for the timely prevention and treatment of equine digestive tract parasitic diseases, and is of great significance for ensuring the health of horse herds and improving breeding efficiency.

Claims

1. A primer set for simultaneously detecting four types of equine gastrointestinal parasites, characterized in that, The primer set includes primer sequences shown in SEQ.ID.NO.1-8, and the four equine gastrointestinal parasites are Parascaris equorum, Strongyloides westeri, Giardia lamblia, and Eimeria spp.

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

3. A kit for simultaneously detecting four types of equine gastrointestinal parasites, 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 gastrointestinal parasites according to claim 3, characterized in that, It also includes a reagent for nucleic acid extraction of the test sample and 2×Universal SYBR qPCR Mix.

5. A method for detecting equine gastrointestinal parasites for non-diagnostic purposes, characterized in that, The method includes the following steps: (1) Extract the total DNA of the test sample; (2) Using the total 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 gastrointestinal parasites 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 upstream and downstream primers of the four gastrointestinal parasites, and 8 μL of the DNA template of the test sample.

7. The method for detecting equine gastrointestinal parasites 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 gastrointestinal parasites for non-diagnostic purposes according to claim 5, characterized in that, Obtain the amplification curve after multiplex PCR amplification, and judge whether there are gastrointestinal parasites according to the Ct value of the amplification curve: If the Ct value of the amplification curve of the test sample > 36 or NoCt, the test result is negative, indicating that there are no above-mentioned gastrointestinal parasites in the test sample; If the Ct value of the amplification curve of the test sample is: 0 < Ct < 36, the test result is positive, indicating that the test sample contains one or more of the above-mentioned gastrointestinal parasites.

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