Fluorescent quantitative PCR (polymerase chain reaction) detection primers and kit for Eimeria media-rabbit

A technology for fluorescent quantitative detection and Eimeria coccidiosis, which is applied in the field of detection, can solve the problems of increasing the difficulty of differential diagnosis for clinical operators, and the morphological characteristics of oocysts are not very obvious, and achieve objective result judgment, high sensitivity, Simple operation effect

Active Publication Date: 2016-03-23
SOUTH CHINA AGRI UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

Rabbit coccidiosis is a mixed infection, and the morphological characteristics of the oocysts of each species are not very obvious, which increases the difficulty of differential diagnosis for clinical operators

Method used

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  • Fluorescent quantitative PCR (polymerase chain reaction) detection primers and kit for Eimeria media-rabbit
  • Fluorescent quantitative PCR (polymerase chain reaction) detection primers and kit for Eimeria media-rabbit
  • Fluorescent quantitative PCR (polymerase chain reaction) detection primers and kit for Eimeria media-rabbit

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Experimental program
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Effect test

Embodiment 1

[0024] Example 1 Primer Design and Screening

[0025] Firstly, the ribosomal ITS sequence of Eimeria mesococcidia isolated from Yulin, Guangxi was amplified by PCR and sequenced, and sequence analysis was performed with the sequence published in GenBank. It was found that the ITS sequence showed relative conservation within the species and The characteristics of large differences among species are suitable as molecular marker sequences for molecular identification between species. Design specific primers in the conserved regions within the species, and refer to the specific primers reported abroad, and screen out the most suitable primers through experimental verification for quantitative experiments.

[0026] Table 1 Design of primers for quantitative PCR of rabbit Eimeria mesodium

[0027]

[0028] Fluorescent quantitative PCR was performed on the sequences designed in Table 1, and the results showed that only primer set 1 could specifically amplify.

[0029] In order t...

Embodiment 2

[0030] The preparation and use method of embodiment 2 kit

[0031] The kit contains stool DNA extraction reagent (QIAampDNAStoolMiniKit), which contains 100 reactions of 140mL ASLBuffer, 1.5mL proteinaseK, 250mL BufferAL, 50mL BufferAW1, 50mL BufferAW2, 10mL BufferAE, 20mL 96%-100% Alcohol; the fluorescent quantitative PCR reaction solution is 100 reactions of SYBRGreenPrimixExTaqII reagent, and the final concentration is 25 pmol / μL of Eimeria medusia specific primers.

[0032] One, the use method of above-mentioned kit, comprises the following steps:

[0033] (1) Extraction of stool DNA:

[0034] Refer to the instructions of QIAampDNAStoolMiniKit, the specific operation steps are as follows:

[0035] a. Weigh 200mg of feces and place it in a 2mL centrifuge tube.

[0036] b. Add 1.4mLASLBuffer and 0.4g glass beads (1mm), shake for 1-2h.

[0037] c. Place the suspension at 70°C for 5min.

[0038] d. Vortex for 15 seconds, then centrifuge the sample at 15,000 rpm for 1 minute...

Embodiment 3

[0062] The sensitivity test of embodiment 3 kits

[0063] Rabbit Eimeria medus 10 6 Make an oocyst DNA stock solution at a ratio of 1:10 to 1:10 6 Doubling dilution, detect with the real-timePCR method that embodiment 2 establishes, analysis sensitivity result sees image 3 , the DNA of at least 10 oocysts can be detected.

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Abstract

The invention relates to the technical field of detection and particularly discloses fluorescent quantitative PCR (polymerase chain reaction) detection primers and kit for Eimeria media-rabbit. The primers are Me-F and Me-R with the sequences shown as SEQ ID NO.1-2; a reaction system and reaction conditions are optimized on the basis of the primers, one fluorescent quantitative PCR kit for quantitative detection of Eimeria media-rabbit on the basis of a fluorescent dye method is developed, the kit operation is simple and programing, the method has high specificity and high sensitivity, and result determination is objective. The method can realize epidemiological investigation and clinical diagnosis of rabbit coccidiosis and provides technical support for diagnosis, prevention and treatment technologies of rabbit coccidiosis and rapid and accurate clinical detection of the rabbit coccidiosis infection condition.

Description

technical field [0001] The invention relates to the technical field of detection, and more specifically relates to a fluorescent quantitative PCR detection primer and a kit for rabbit Eimeria mesodium. Background technique [0002] Rabbit coccidiosis is one of the most common protozoal diseases caused by Eimeria rabbits parasitizing in the liver and bile ducts and intestinal epithelial cells of rabbits. The disease is serious, popular, and has a high infection rate. It is extremely harmful to the rabbit industry and causes huge economic losses every year. It is distributed worldwide. It is reported that the infection rate of young rabbits aged 1 to 3 months can reach 100%. The mortality rate of young rabbits after the disease is as high as about 70%, and infection reports have also been reported in many areas of my country. There are as many as 11 species of pathogenic species of the disease, namely Eimeria stiedai (Eimeriastiedai), Eimeria perforans (Eimeria perforans), Eim...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/6893C12Q2563/107
Inventor 林瑞庆许家园翁亚彪胡会朋李国良何茜吕梦娜
Owner SOUTH CHINA AGRI UNIV
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