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Method for detecting Echinococcus multilocularis from fox excrement

A technology of Echinococcus multilocularis and fox, applied in the field of identification of Echinococcus multilocularis pathogens, can solve the problems of low sensitivity and specificity, danger of infecting people and the surrounding environment, and achieve high accuracy and low cost Effect

Inactive Publication Date: 2015-11-04
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, existing detection methods such as conventional etiological detection methods (arecoline catharsis method and autopsy method), ELISA detection method, LAMP detection technology, etc. (Dinkel A, et al. Detection of Echinococcus multilocularis in the definitive host: coprodiagnosis by PCR as an alternative to Necropsy[J].J Clin Microbiol,1998,36(7):1871-1876.; Eckert J.Predictive values ​​and quality control of techniques for the diagnosis of Echinococcus multilocularis in definitive hosts.Acta Trop ,2003,85(2):157-163.; Ni XM.Loop-Mediated Isothermal Amplification(LAMP) assay for the identification of Echinococcus multilocularis infections in canine definitive hosts.Particle and Fiber Toxicology,2014,7(1):35 -42.) There are certain shortcomings, the sensitivity and specificity are low, and the routine detection method also has the risk of infecting people and the surrounding environment

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  • Method for detecting Echinococcus multilocularis from fox excrement
  • Method for detecting Echinococcus multilocularis from fox excrement
  • Method for detecting Echinococcus multilocularis from fox excrement

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Embodiment 1

[0025] 1 Collection of Tibetan fox feces samples: Tibetan fox feces samples were collected from the home range of 7 radio-tracked Tibetan foxes (Yunbogou, Erdoma Township, Shiqu County, Sichuan Province (N 33°10′, E 97 °39′)), all samples were stored in 70% ethanol, and for safety reasons, they were frozen at -80°C for more than 3 weeks before the experiment.

[0026] 2 Enrichment of feces eggs: Take 2g of feces and put them in a 150ml beaker, add 100ml of deionized water. Water bath at 80°C for 10 minutes, mince with scissors, and mix well. Filter the turbid liquid with a 100-mesh filter, store the filtrate in a 50ml test tube, pour the filtrate on the double-layer sterilized gauze laid flat on the plate, squeeze the gauze several times, let the turbid liquid seep out, and collect it In a 50ml test tube. Centrifuge at 3600g for 30min, discard the supernatant.

[0027] 3. Fragmentation of worm egg embryo membrane: add 600ul ASL, mix it with the precipitate with a pipette ti...

Embodiment 2

[0032] We designed a nested PCR method to screen for the presence of Echinococcus multilocularis genetic material in fox feces. The outer primers were the general primers (F1, R1) used to amplify the partial sequence of mitochondrial DNA cytochrome oxidase I subunit gene (COI), with a length of 874bp. The inner primer was designed with Primer Premier 5.0 software, and the product length was 243bp.

Embodiment 3

[0033] Effect evaluation of embodiment 3 nested PCR

[0034] We randomly selected 72 samples from a total of 184 stool samples collected in 2010 to evaluate the established fecal DNA nested PCR diagnostic method. Among them, the DNA of 48 samples was successfully extracted, and after species identification, all of them came from Tibetan foxes. After enrichment and crushing of tapeworm eggs in fecal samples ( figure 1 ), we used nested PCR and Xiao(Xiao N, Nakao M, Qiu JM, Budke CM, Giraudoux P, Craig PS, Ito A(2006a) Short Report: Dual infection of animal hosts with different species of Echinococcus in the eastern Qinghai -Tibetan Plateau region of China.Am J Trop Med Hyg,75:292-294.) Two methods of PCR-RFLP were established to screen the Em genetic material in fox feces, and found that the detection results of the two methods were significantly different <0.001, Wilcoxon sign test for paired samples). Among the 48 stool samples, nested PCR detected 9 (19%) samples infected...

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Abstract

The invention discloses a method for detecting Echinococcus multilocularis pathogen from fox excrement. The method includes: placing the collected Tibetan fox excrement sample into 70% ethanol, and performing cryopreservation at minus 80 DEG C for more than three weeks; using double-layer sterilizing gauze to enrich excrement sample Echinococcus multilocularis eggs, using a tissue crusher to crush egg embryonic membranes in a mechanical oscillation manner, using an excrement DNA extracting kit to extract the DNA of the Echinococcus multilocularis egg in the excrement sample, and using nest PCR amplification to detect the Echinococcus multilocularis eggs. The method is simple to operate, capable of fast detecting whether Echinococcus multilocularis infection exists in the fox excrement or not, high in sensitivity and high in specificity.

Description

technical field [0001] The invention belongs to the field of molecular biology detection and relates to the identification of Echinococcus multilocularis pathogens in stool samples. Background technique [0002] Alveolar echinococcosis (AE) is a fatal zoonotic parasitic disease caused by Echinococcus multilocularis (Em). Because there is no specific treatment for the disease, it is called parasitic cancer. AE is mainly prevalent in pastoral and semi-agricultural and semi-pastoral areas in the northwest of my country. Among them, the eastern pastoral areas of the Qinghai-Tibet Plateau are the most severely affected areas in the world. At present, AE has become one of the most serious public health safety problems in plateau pastoral areas in my country. [0003] Tibetan fox (Vulpes ferrilata) and domestic dogs are the main final hosts of Em in pastoral areas in eastern Tibet. However, due to the wide range of free activities of domestic dogs and close contact with humans, e...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888
Inventor 蒋韦斌王正寰王小明
Owner SHANGHAI NORMAL UNIVERSITY
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