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A kind of multiple dpo-pcr primer combination and method for detecting Trichinella spiralis, Toxoplasma gondii and Schistosoma

A DPO-PCR, primer combination technology, applied in microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of complex system, low stability, limited use, etc., to achieve high throughput, Good specificity and improved detection efficiency

Inactive Publication Date: 2020-08-04
海南出入境检验检疫局检验检疫技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the addition of multiple pairs of primers in the PCR reaction system, the system is complex and the stability is not high, which limits its use

Method used

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  • A kind of multiple dpo-pcr primer combination and method for detecting Trichinella spiralis, Toxoplasma gondii and Schistosoma
  • A kind of multiple dpo-pcr primer combination and method for detecting Trichinella spiralis, Toxoplasma gondii and Schistosoma
  • A kind of multiple dpo-pcr primer combination and method for detecting Trichinella spiralis, Toxoplasma gondii and Schistosoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Multiplex DPO-PCR method for detecting Trichinella spiralis, Toxoplasma gondii and Schistosoma

[0029] 1. Design of Multiplex DPO-PCR Primer Combinations

[0030] According to the conserved regions of Trichinella spiralis ATP6 target gene, Toxoplasma gondii P22 target gene and Schistosoma japonicum 28S rRNA, the following multiplex DPO-PCR primer combinations for detecting the conserved regions of Trichinella spiralis, Toxoplasma gondii and Schistosoma japonicum were respectively designed (I in the primer sequence xanthine):

[0031] Trichina-F: 5'-TCTCCCTACTCAGATACAACTGAATIIIIIIACAGCCAA-3' (SEQ ID NO: 1)

[0032] Trichina-R: 5'-GGATTTATGTGTTTTTTTGTGTGTGTTIIIIITTCTGGTC-3' (SEQ ID NO: 2)

[0033] Toxo-F: 5'-CGGCGCAACGAAGACTGTTGIIIIICCCTCCAGT-3' (SEQ ID NO: 3)

[0034] Toxo-R: 5'-ACCTGCTTCGGCAACGCACTTIIIIIIAGAGAACC-3' (SEQ ID NO: 4)

[0035] Schistosome-F: 5'-TGAGATACCACAAAAGGTGTTGGIIIIICCAGACAGC-3' (SEQ ID NO: 5)

[0036] Schistosome-R: 5'-GGGCTGCGATCTAC...

Embodiment 2

[0052] Example 2. Sensitivity detection of multiple DPO-PCR primer combinations

[0053] 1. Extraction of DNA

[0054] According to the instructions of the kit, the gene DNA of Trichinella spiralis, Toxoplasma gondii and Schistosoma japonicum was extracted. The obtained genomic DNA was mixed and diluted 10 times, and the genomes of Trichinella spiralis, Toxoplasma gondii and Schistosoma japonicum were prepared at concentrations of 18ng / μL, 1.8ng / μL, 0.18ng / μL, 0.018ng / μL and 0.0018ng / μL, respectively. DNA.

[0055] 2. Multiplex DPO-PCR amplification

[0056] Adopt the method for detecting Trichinella spiralis, Toxoplasma gondii and Schistosoma in the step 2 of embodiment 1, carry out multiple DPO-PCR amplification with the genomic DNA of following 5 groups respectively as template, obtain multiple DPO-PCR amplification products respectively:

[0057] Group 1: Genomic DNA (1 μL) of 18 ng / μL Trichinella spiralis, Toxoplasma gondii and Schistosoma japonicum was used as templat...

Embodiment 3

[0065] Example 3. Detection of Annealing Temperature Sensitivity of Multiplex DPO-PCR Primer Combinations

[0066] 1. Extraction of DNA

[0067] Genomic DNA of Trichinella spiralis, Toxoplasma gondii and Schistosoma japonicum was extracted according to the kit instructions.

[0068] 2. Multiplex DPO-PCR amplification

[0069] Using the Trichinella spiralis, Toxoplasma gondii and Schistosoma genomic DNA obtained in step 1 as a template, using conventional primers (as shown in Table 1) or the method for simultaneously detecting Trichinella spiralis, Toxoplasma gondii and Schistosoma in step 2 of Example 1, using different The annealing temperatures (48.3°C, 49.5°C, 51.4°C, 53.7°C, 56.4°C, 59.1°C, 61.7°C, 64.1°C, 66.1°C, 67.4°C, 68°C) were used for PCR amplification, and the rest of the reaction conditions were unchanged, respectively Obtain the PCR amplification product.

[0070] Table 1. Sequences of conventional primers for amplifying 28S rRNA of Schistosoma japonicum, ATP6...

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Abstract

The invention provides a multiple DPO-PCR (dual priming oligonucleotide-polymerase chain reaction) primer combination for detecting trichinella spiralis, toxoplasma gondii and schistosome. The sequences of the primer combination are as shown in SEQ ID NO:1 to SEQ ID NO:6 correspondingly. The invention also provides a multiple DPO-PCR method for detecting the trichinella spiralis, the toxoplasma gondii and the schistosome. By adopting the method, qualitative detection on the muscular tissue or viscera sample of animals suffering from diseases can be realized. Experiments indicate that the detection method provided by the invention has high specificity, high flux and high speed, the detection efficiency is improved, and a more effective method is provided for detecting three animal origin parasitic zoonoses of the trichinella spiralis, the toxoplasma gondii and the schistosome.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a combination of primers and a method for simultaneously detecting Trichinella spiralis, Toxoplasma gondii and Schistosoma japonicum based on multiplex DPO-PCR. Background technique [0002] Trichinella spiralis is a serious food-borne zoonotic parasitic nematode, which can infect more than 150 mammals including humans and is distributed worldwide. Trichinellosis infection comes from ingestion of raw or undercooked pork or other animal meat containing trichinella cysts, and human infection with trichinellosis can cause death. It is a zoonotic parasitic disease that seriously endangers public health safety , so it is a mandatory item in meat hygiene inspection. [0003] Toxoplasma gondii is an obligate intracellular parasitic protozoan that can infect almost all nucleated cells of warm-blooded animals, and is a zoonotic parasitic disease that is widesprea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6893C12Q1/686C12Q1/04C12N15/11C12R1/90
CPCC12Q1/686C12Q1/6888C12Q1/6893C12Q2600/16C12Q2537/143C12Q2525/161
Inventor 李丹丹徐义刚张体银安微杨俊兴邱索平王绥家陈文慧陈平亚
Owner 海南出入境检验检疫局检验检疫技术中心
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