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Polymerase chain reaction (PCR) detection method of porcine eperythrozoon

A technology of Eperythrozoon porcine and detection method, which is applied in the direction of biochemical equipment and methods, microbe measurement/inspection, etc., can solve the problems of non-specific repeatability, long time-consuming, high false positive rate, etc., and achieve high specificity, Low price, high sensitivity effect

Inactive Publication Date: 2014-06-18
QINGDAO ZHONGREN PHARMA
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Problems solved by technology

Clinically, the diagnostic method of the disease is mainly based on direct microscopic examination, including fresh blood pressure film and smear staining, but the false positive rate is high. Animal experiment method is the means of diagnosis, but it takes too long and expensive. Serum The clinical methods include indirect hemagglutination test, complement fixation test or enzyme-linked immunosorbent assay, etc., because the antibody of Eperythrozoon changes greatly, these methods are not suitable for the diagnosis of the disease, although PCR method is used to diagnose pigs in recent years Reports of Eperythrozoonosis are not uncommon, but in the actual application process, some will still show non-specificity and poor repeatability. In addition, the EB used in PCR detection is a harmful substance, so it is not an ideal method.
Real-time fluorescent quantitative PCR is a new nucleic acid technology developed in recent years. It has the advantages of real-time, sensitive, fast, and safe. Quantitative PCR detection report of porcine Eperythrozoon

Method used

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Embodiment

[0023] The porcine Eperythrozoon PCR detection method of the present embodiment comprises the following steps:

[0024] (1) Isolation and purification of pathogens, strictly aseptically collect 1ml of blood samples from 10 suspicious pigs, store at 4°C or freeze, add 3 times 0.01mol / L PBS pH7.4 to each collected blood sample for washing, 2000r / Min centrifuge for 10 min, remove the supernatant, add 1 times PBS to the precipitate to dilute, bathe in 56°C water for 3 min, immediately centrifuge at 2000r / min for 10min, discard the precipitate, take the supernatant in another centrifuge tube, centrifuge at 15000r / min for 2h , remove the supernatant, add 1ml of normal saline to the precipitate to dilute, and store at 4°C or -20°C for later use;

[0025] (2) For the extraction of Eperythrozoon porcine DNA, according to the instructions of the Genomic DNA purification kit, extract the Eperythrozoon porcine template from the purified Eperythrozoon porcine pathogen and the standard pos...

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Abstract

The invention discloses a polymerase chain reaction (PCR) detection method of porcine eperythrozoon, and the method successively comprises the steps of pathogen separation and purification, porcine eperythrozoon DNA extraction, primer design and synthesis, ordinary PCR detection, positive plasmid standard substance preparation and real-time fluorescent quantitative PCR detection. Compared with IHA (indirect hemagglutination assay), ELISA (enzyme-linked immuno sorbent assay) and other detection methods, the method has the advantages of high specificity, high sensitivity, high accuracy and reliability, can fast, simply, reliably and accurately detect eperythrozoon in a sample, and can also be used for epidemiological investigation and curative effect detection of eperythrozoonosis.

Description

[0001] technical field [0002] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting eperythrozoonosis through real-time fluorescent quantitative polymerase chain reaction (PCR). [0003] Background technique [0004] Eperythrozoonsis (Eperythrozoonsis) is a zoonotic disease with anemia, jaundice and fever as the main clinical symptoms caused by Eperythrozoon (EH) parasitizing on the surface of human and animal red blood cells, plasma and bone marrow. . Belongs to Rickettsiales (Rickettsiales), Anaplasmata-ceae (Anaplasmata-ceae), Eperythrozoon (Eperythrozoon). Most of them are single or clustered on the surface of red blood cells, in the form of chains or scales, and a small amount of them are free. [0005] Porcine Eperythrozoon is generally ring-shaped, with a diameter of 0.8-2.5um, and also spherical and rod-shaped. It is sensitive to drying and chemical drugs. 0.5% carbolic acid can kill it after 3 hours at 37°C. G...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06
CPCC12Q1/686C12Q1/04
Inventor 张述智夏伟朱绍辉张浩王晓丽徐权汗李之详许团辉
Owner QINGDAO ZHONGREN PHARMA
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