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Neospora caninum specific PCR detection kit, and preparation method and application thereof

A technology of Neospora caninum and detection kit, which is applied in biochemical equipment and methods, microbe measurement/inspection, DNA/RNA fragments, etc. It can solve the problems that the detection of the disease cannot meet the actual needs and the quarantine diagnosis lags behind. Achieve great social significance, high accuracy, and broad application prospects

Active Publication Date: 2020-05-26
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The quarantine diagnosis of canine neosporidiosis at home and abroad is still relatively lagging behind, and the detection of the disease is far from meeting the actual needs

Method used

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  • Neospora caninum specific PCR detection kit, and preparation method and application thereof
  • Neospora caninum specific PCR detection kit, and preparation method and application thereof
  • Neospora caninum specific PCR detection kit, and preparation method and application thereof

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Effect test

Embodiment 1

[0027] According to the principle of PCR amplification, the present invention designs and synthesizes a group of PCR primers for amplifying the specific gene sequence SEQID NO.1 of Neospora caninum. The synthesis of the primers is entrusted to Beijing Ruiboxing Biotechnology Co., Ltd. After primer synthesis, use sterilized distilled water to make 100mM stock solution (stock solution), and then use ddH 2 O was diluted to 10 mM as a working solution. The primer sequences are as follows:

[0028] Upstream primer F: 5'-CGCACAAGAAACCGAGGAA-3' (SEQ ID NO.2)

[0029] Downstream primer R: 5'-GAAGGCGAGAAGCCCAAGT-3' (SEQ ID NO.3)

Embodiment 2

[0031] kit assembly

[0032] 1) 10×PCR reaction solution: 20 μL of 300 mM dNTPs (purchased from TAKARA, Japan), 2.42 mg of Tris (purchased from Pragma, USA), 7.45 mg of KCl (purchased from Sigma, USA), MgCl2 (purchased from Sigma, USA) 0.258mg, 200U Taq enzyme (purchased from Japan TAKARA company) 20μL, add 150μL ddH 2 O, adjust the pH to 8.5 with HCl, use ddH 2 O to dilute to 200 μL.

[0033]2) Preparation of positive control DNA: the positive control DNA is Neospora caninum genomic DNA, and the cultured Neospora caninum is counted with a cell counting plate and counted with 10 7 Genomic DNA was extracted from each Neospora caninum and diluted to 100 μL with water.

[0034] 3) Assembly of the kit:

[0035] Assemble the kit as follows (the entire operation requires a sterile environment):

[0036] 10×PCR reaction solution 200μL

[0037] 10mM upstream primer F 50μL

[0038] 10mM downstream primer R 50μL

[0039] Positive control DNA 100μL

[0040] wxya 2 O 2mL

Embodiment 3

[0042] The using method of kit of the present invention is as follows:

[0043] Take the sample to be tested, extract genomic DNA according to conventional methods, and perform PCR amplification with the reagents in this kit. The PCR reaction sample loading system is as follows, the total reaction system is 20 μL (the amount of sample DNA is determined according to the concentration of the extracted genomic DNA, and finally with ddH 2 O to make up 20 μL; positive control volume is 1 μL):

[0044] 10×PCR reaction solution: 2μL

[0045] Determine the sample DNA according to the amount of extracted DNA

[0046] Upstream primer F (10mM): 1μL

[0047] Downstream primer R (10mM): 1μL

[0048] wxya 2 O: Make up to 20 μL

[0049] Add the negative and positive controls to the marked tubes respectively, then add the samples one by one and mark them, and place them in the PCR machine. The PCR reaction conditions were: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30...

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Abstract

The invention discloses a Neospora caninum specific PCR detection kit, and a preparation method and an application thereof. The specific gene sequence of the Neospora caninum is represented by SEQ IDNO.1. The specific gene sequence has five copies in a Neospora caninum genome. An amplification primer of the specific gene sequence is further designed, and the Neospora caninum PCR detection kit isconstructed, and is applied to clinical detection and screening of animal Neospora caninum infection to accurately and quickly diagnose female animal abortion pathogens caused by Neospora caninum infection and investigate animal population Neospora caninum infection.

Description

technical field [0001] The invention relates to the technical field of animal inspection and quarantine, in particular to a Neospora caninum-specific PCR detection kit, its preparation method and application. Background technique [0002] Neospora caninum (Neospora caninum) belongs to the intracellular parasitic protozoa of the subphylum Apicomplexa, which can infect a variety of animals, and can cause neuromuscular system dysfunction and reproductive disorders in pregnant animals in cattle, dogs and other animals. In order to effectively detect N. caninum infection, researchers have established a variety of serological and immunological detection methods, including indirect immunofluorescence assay (IFAT), enzyme-linked immunosorbent assay (ELISA), and modified agglutination test (NAT). Among them, ELISA is the most widely used to detect N. caninum infection, but it is not effective for early infection and infection in calves within 5 months of age. PCR amplification is cu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6893C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6893
Inventor 刘群杨聪山刘晶许建海
Owner CHINA AGRI UNIV
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