Loop-mediated isothermal amplification detection method of E. wenyoni

A technology of epierythropoiesis and isothermal amplification, which is applied in the field of parasite molecular detection, can solve the problems of high requirements for detection personnel and high detection costs, and achieve the effect of ensuring high specificity, simple detection and strong specificity.

Inactive Publication Date: 2011-01-19
HUAZHONG AGRI UNIV
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] PCR amplification of the 16S rRNA gene of Eperythrozoon bovis at the molecular level can timely and accurately identify pathogens, but ordinary PCR, nested PCR and fluorescent quantitative PCR with higher sensitivity must rely on expensive precision instruments. High testing costs and high technical requirements for testing personnel cannot be carried out in basic laboratories with poor conditions

Method used

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  • Loop-mediated isothermal amplification detection method of E. wenyoni
  • Loop-mediated isothermal amplification detection method of E. wenyoni
  • Loop-mediated isothermal amplification detection method of E. wenyoni

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1: Carrying out the detection of Eperythrozoon Wennneri on a blood sample

[0044] 1. Extraction of the total DNA of the bovine blood sample: extract the DNA in the bovine blood sample with the blood / cell / tissue genomic DNA extraction kit (spin column type) (purchased from TIANGEN company, catalog number: DP304-03, operate according to the kit instructions) total DNA.

[0045] 2. Loop-mediated isothermal amplification:

[0046] A loop-mediated isothermal amplification (LAMP) reaction system was established as follows: 2.5 μL of 10-fold volume of BstDNA polymerase buffer (the composition is 0.2M Tris-HCL, 0.1M (NH 4 ) 2 SO 4 , 0.1M KCL, 20Mm MgSO 4 , 1% TritonX-100), 2mM MgCl 2 , 0.6M betaine (betaine), 0.4mM dNTPs, 0.08μM each of the outer primer F3B3, 0.6μM each of the inner primer FIP BIP, 0.28μM each of the loop-promoting primer LF LB, 8U Bst DNA polymerase, 5μL DNA template, with sterile Make up to 25 μL with double distilled water. Wherein the pos...

Embodiment 3

[0046] A loop-mediated isothermal amplification (LAMP) reaction system was established as follows: 2.5 μL of 10-fold volume of BstDNA polymerase buffer (the composition is 0.2M Tris-HCL, 0.1M (NH 4 ) 2 SO 4 , 0.1M KCL, 20Mm MgSO 4 , 1% TritonX-100), 2mM MgCl 2 , 0.6M betaine (betaine), 0.4mM dNTPs, 0.08μM each of the outer primer F3B3, 0.6μM each of the inner primer FIP BIP, 0.28μM each of the loop-promoting primer LF LB, 8U Bst DNA polymerase, 5μL DNA template, with sterile Make up to 25 μL with double distilled water. Wherein the positive control is the constructed bovine Eperythrozoon positive plasmid (the specific construction method will be described in Example 3: Sensitivity Test of the Eperythrozoon Wenneri LAMP Detection Method), and the negative control is sterilized double distilled water; 95°C After pre-denaturation for 5 minutes, add 8U of Bst DNA polymerase to the above reaction system except for the enzyme, then place it at 63°C for 50 minutes, and then react...

Embodiment 2

[0050] Example 2: Specificity Test of Bovine Eperythrozoon Bovine LAMP Detection Method

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Abstract

The invention belongs to the technical field of detection of parasite molecules, and in particular relates to a quick loop-mediated isothermal amplification detection method of E. wenyoni, which comprises the following steps: (1) designing three pairs of specific primers; (2) extracting total DNA of a blood sample; and (3) carrying out loop-mediated isothermal amplification reaction and analyzing the amplification product. By imaging on a gel imaging system and observing the result, E. wenyoni is positive if the amplification product is a stepped pattern formed by zones of different sizes; or SYBR Green I is added to the final product of amplification, and by visually observing the result under natural light, the E. wenyoni is positive if the amplification product turns to green yellow, and the E. wenyoni is negative if the amplification product is reddish brown. The invention has strong specificity and can be used for discriminating common blood protozoa, the sensitivity is high and is 10-100 times higher than that of common PCR, the detection is simple and quick, only one water bath is needed, and the result can be obtained in 1 hour.

Description

technical field [0001] The invention belongs to the technical field of parasite molecular detection, and in particular relates to a rapid loop-mediated isothermal amplification detection method of Eperythrozoon wennerii. technical background [0002] Eperythrozoonosis is caused by the obligate blood parasite Eperythrozoon, and is a disease characterized by anemia, jaundice, and fever. Some people at home and abroad have called it icteric anemia, anaminidosis, red animal disease and erythroderma, etc. 【1】 . Since Schilling and Dingen discovered the sphagoid haemozoa in rodents in 1928, scientists have successively discovered the existence of the sphagoid haemozoa in different animals. Among them, in 1934, Adler et al. discovered a microorganism similar in shape to the spheroid hematocides in cattle, and named it E. wenyoni. [0003] The classification of Eperythrozoon has always been controversial. In 1974, the eighth edition of "Berger's Bacterial Identification Manual" ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 宋淇淇王利霞赵俊龙周艳琴胡敏贺兰徐林海张文杰张庆丽
Owner HUAZHONG AGRI UNIV
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