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Method for detecting three kinds of protozoa in duck manure by using multiple PCR

A multiplex, protozoan technology, applied in the field of PCR detection, to achieve broad application prospects and good specificity

Inactive Publication Date: 2010-12-15
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far there is no multiplex PCR method to detect or diagnose the mixed infection of duck intestinal protozoa

Method used

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  • Method for detecting three kinds of protozoa in duck manure by using multiple PCR
  • Method for detecting three kinds of protozoa in duck manure by using multiple PCR
  • Method for detecting three kinds of protozoa in duck manure by using multiple PCR

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0047] Experimental Example 1 Single Item PCR Specificity Test

[0048] The extracted genomic DNA of three kinds of protozoa was used as the amplification template of single PCR, and DNA control groups of various kinds of protozoa were set up at the same time. The DNA control group during the specific PCR amplification of Duck Cyclospora is: Eimeria tenella, Cryptosporidium beineri and Echinococcus philaidei, and the DNA control group during the specific PCR amplification of Echinococcus philaidei is: Eimeria tenella, Eimeria hemiformis and Cryptosporidium beinii, and the DNA control group for the specific PCR amplification of Cryptosporidium beinii in ducks are: Cryptosporidium parvum, Wennella philaisi and duck Cyclospora. The designed species-specific primers were used for PCR amplification, and the reaction system and PCR amplification conditions were as follows.

[0049] (1) Duck Cyclospora-specific PCR

[0050] reaction system:

[0051] 10×buffer 2.5μL

[0052] MgCl...

experiment example 2 3

[0092] Experimental Example 2 Three pairs of primer concentration preliminary test

[0093] Based on the single specific PCR reaction, the DNA templates of the three protozoa were mixed, and three pairs of specific primers were used to establish a multiplex PCR detection method, and the optimal reaction conditions were studied. In order to understand the interaction among the primers when 3 pairs of primers simultaneously amplify their target genes, the final concentration of the primers is set to 1 μM in advance, and the amplification is performed simultaneously according to the following reaction system and reaction conditions.

[0094] (1) Reaction system of multiplex PCR preliminary test

[0095] Mixture components volume (unit: μL)

[0096] 10×PCR Buffer 2.5

[0097] dNTPs (2.5mM) 2.0

[0098] Mg 2+ (25mM) 2.0

[0099] Ex-Taq enzyme (5U / μL) 0.2

[0100] 6 protozoa primers (25nmol / L) each 1.0

[0101] 3 kinds of DNA templates, 2.0 each

[0102] wxya 2 O Add to 25....

experiment example 3

[0106] Determination of Experimental Example 3 Optimum Reaction Conditions for Multiplex PCR

[0107] Optimization of primer concentration:

[0108] According to the results of the preliminary experiment on the concentrations of the three pairs of primers, in the reaction system, keep the concentrations of the primers CB1 / CB2 unchanged, adjust the concentrations of the other two pairs of primers, and gradually increase the addition amount to: 1.2μM, 1.4μM, 1.6μM, 1.8μM, 2.0μM, for multiplex PCR amplification. The results showed that when the concentration of primer CD1 / CD2 was 1.6 μM, the amplification effect was the best; however, the amplification effect of primer WF1 / WF2 was slightly poor, and the concentration of primer WF1 / WF2 needed to be optimized. The concentrations of primers CB1 / CB2 and CD1 / CD2 were fixed at 1.0 μM and 1.6 μM, and the concentrations of primers WF1 / WF2 were gradually increased to optimize the concentration of WF1 / WF2 primers in multiplex PCR. The re...

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Abstract

The invention relates to the field of PRC detection, in particular to a method for detecting three kinds of protozoa in duck manure by using multiple PCR, wherein the protozoa include Cryptosporidium baileyi, Cyclospora and Wenyonella philiplevinei. The detection method of the invention comprises: designing three pairs of specific premiers according to the ITS-1 gene of the Cryptosporidium baileyi, the 18S rRNA gene of the Wenyonella philiplevinei and the ITS-1+ fragment of the Cyclospora of ducks respectively; and quickly and accurately detecting the oocysts of the Cyclospora, the Cryptosporidium baileyi and the Wenyonella philiplevinei in the duck manure by multiple PCR reactions to identify and diagnose the Cryptosporidiosis, Cyclospora disease and coccidiosis of ducks. The detection method of the invention can detect the three kinds of protozoa at the same time, has high sensitivity and specificity, is more quick and economic than conventional PCR and can be used for the identification and diagnosis of duck intestinal protozoa disease, molecular epidemiological survey and waterborne Cryptosporidiosis and Cyclospora disease outbreak risk evaluation.

Description

technical field [0001] The invention relates to the field of PCR detection, in particular to a method for detecting three kinds of protozoa in duck feces by using multiple PCR. Background technique [0002] A variety of parasitic protozoa can be stored and infected in the duck intestines. These protozoa are excreted with feces and can be transmitted among ducks, and some can even infect humans. This not only affects their own survival and the development of the poultry industry. Serious hazards can also pose a threat to human health. Among them, Echinococcus philaisi is the main pathogen causing coccidiosis in ducks, and Cryptosporidium bellini and Cyclosporidium can cause zoonotic parasitic diseases, which pose a serious threat to the breeding industry and human health. [0003] Because duck intestinal parasites can cause common digestive tract symptoms clinically, and often present as mixed infections, it is difficult to distinguish them through clinical diagnosis. The c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 李国清程家林岳彩玲高振永刘霞朱海波
Owner SOUTH CHINA AGRI UNIV