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Primer and detection kit for detecting seven types of chicken eimeria tenella

A technology of Eimeria gallinarum and Eimeria gallinarum, which is applied in the field of primers and detection kits for detecting seven kinds of Eimeria gallinarum, can solve problems such as complexity and inaccurate diagnosis and identification techniques, and achieve results Judgment is objective, highly sensitive, and simple to operate

Active Publication Date: 2013-12-18
ZHAOQING INST OF BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the inaccurate and complex defects of the prior art coccidia species diagnosis and identification technology, the present invention provides a primer for detecting seven Eimeria chicken species

Method used

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  • Primer and detection kit for detecting seven types of chicken eimeria tenella
  • Primer and detection kit for detecting seven types of chicken eimeria tenella
  • Primer and detection kit for detecting seven types of chicken eimeria tenella

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Design of 7 pairs of specific primers: In this study, the ribosomal DNA of Eimeria gallinarum isolates from different endemic areas was analyzed for genetic variation, and it was found that the ribosomal IGS sequence and ITS sequence were different in different Eimeria strains of the same species. The differences between insects are very small, but the differences between different species are very large. The inventor designs 7 kinds of specific primers according to the IGS and ITS specific sequences of 7 kinds of Eimeria as following table 1:

[0085] Table 1 Specific primers for fluorescent quantitative PCR of seven Eimeria species

[0086]

Embodiment 2

[0088] Fluorescent quantitative PCR kit for quantitative detection of 7 species of Eimeria

[0089] The kit includes the stool DNA extract to be detected; the SYBR GREEN I reaction mixture of 400 reactions; the final concentration of 7 kinds of Eimeria specific primers shown in Example 1 of 50 pmol / μL; sterile water. The SYBR GREEN I reaction mixture is a reagent purchased from TaKaRa Company, and the SYBR GREEN I reaction mixture contains TaKaRa Ex Taq HS, dNTP Mixture, Mg 2+ and SYBR ? Green I.

[0090]

[0091] The fecal DNA extract to be tested includes: 140 mL of ASL Buffer for 50 reactions; 1.4 mL of proteinase K; 33 mL of Buffer AL; 19 mL of Buffer AW1; 13 mL of Buffer AW2; 12 mL of Buffer AE; 96% to 100% alcohol; 100 mL of 10% sodium hypochlorite. Among them, ASL Buffer, proteinase K, Buffer AL, Buffer AW1, Buffer AW2 and Buffer AE are derived from the DNA extraction kit of Tiangen. Of course, the extraction of the DNA of the stool sample to be detected in the pr...

Embodiment 3

[0117] Kit specificity test described in embodiment 2

[0118] Using 5 μL of Eimeria chicken DNA from 7 species of Eimeria chickens that has been verified for DNA validity as a template, the 7 pairs of primers were subjected to specific fluorescence quantitative PCR amplification according to the reaction conditions of the kit, and a blank was set at the same time (the template for the blank control was sterile water). The reaction system was: 8 μL of SYBR GREEN I reaction mixture, 1 μL of each primer, 10 μL of double distilled water, and 5 μL of template DNA.

[0119] Fluorescent quantitative PCR amplification conditions are:

[0120]

[0121] The results show that: seven pairs of primers can specifically amplify seven kinds of Eimeria (see Figure 1-7 ).

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Abstract

The invention belongs to the technical field of biological detection and particularly discloses a primer and a detection kit for detecting seven types of chicken eimeria tenella. The primer is obtained by designing IGS (Intergenic Spacer) and ITS (Internal Transcribed Spacer) specific sequences of the seven types of eimeria tenella, namely eimeria tenella, eimeria necatrix, eimeria maxima, eimeria acervulina, mild eimeria tenella, eimeria praecox and eimeria brunette. A fluorescence quantitative PCR (Polymerase Chain Reaction) kit prepared by the seven pairs of primers can be used for accurately, objectively and quantitatively detecting the seven types of chicken eimeria tenella; the detection kit has very high specificity and sensitivity.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a primer and a detection kit for detecting seven kinds of Eimeria gallinarum. Background technique [0002] Chicken coccidiosis is a common parasitic disease caused by Eimeria coccidiosis. The disease is extremely harmful, and the global loss caused by coccidiosis infection is about 3 billion U.S. dollars every year. It is one of the diseases that cause the most serious economic losses among chicken diseases. There are 7 kinds of Eimeria chickens that have been discovered and recognized, namely Eimeria tenabilis, Eimeria poisonous, Eimeria giant, Eimeria pileus, and Eimeria placidum. Coccidia, Eimeria precoccus and Eimeria brucei, all coccidia are somewhat pathogenic. The disease is widely distributed, and the infection often manifests as two or more mixed infections, with a morbidity rate as high as 50% to 70%, a mortality rate of 20% to 30%, and severe cases as h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 翁亚彪刘国昌林瑞庆袁子国吴林林王新秋段雯方
Owner ZHAOQING INST OF BIOTECHNOLOGY CO LTD
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