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Field mouse babesia RPA molecular detection method

A technology of Babesia and detection methods, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of high detection costs, achieve fast and time-saving effects, prevent background noise, and have flexible volumes

Active Publication Date: 2018-09-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Specific amplification of Babesia microti genes based on the molecular level, but ordinary PCR, nested PCR and fluorescent quantitative PCR must rely on expensive instruments, high detection costs, and require high technology for testing personnel, etc. place with poor conditions

Method used

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  • Field mouse babesia RPA molecular detection method
  • Field mouse babesia RPA molecular detection method
  • Field mouse babesia RPA molecular detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: Carry out the detection of Babesia microti to blood sample

[0031] 1. Extraction of total DNA from blood samples of BALB / c mice: The total DNA in blood samples was extracted using QIAamp DNA Mini Kit (Qiagen, Germany) according to the instructions of the kit.

[0032] 2. Gene selection

[0033] Analyzing more than 10 articles on the detection of Babesia microti, it was found that most of them used 18S rRNA as the target sequence for detection, but it was difficult to synthesize RPA primers because of its short specific region. We selected the mitochondrial gene and compared the full-length mitochondrial length (11109bp) of different strains published in GenBank, and then compared the sequences with Bioedit software. gene) showed high specificity to protozoa and various hosts. Therefore, the highly specific segment of the COX I gene of Babesia microti was selected as a candidate template for primer design.

[0034] 3. Determining the target sequence

...

Embodiment 2

[0045] Embodiment 2: Babesia vole RPA detection method specificity test

[0046] As shown in Table 1, the Theileria buffalo, Babesia canis, Babesia orientalis and Toxoplasma gondii strains used in the specificity test were isolated and preserved by the animal parasite laboratory where the applicant works Insect strain. Plasmodium falciparum DNA was donated by Chongqing Third Military Medical University. All strains were preserved in frozen anticoagulated form. In addition, human, dog, buffalo and BALB / c mouse gDNA were all provided by the applicant's animal parasite laboratory.

[0047] Table 1 Specificity test for RPA detection of Babesia vole

[0048]

[0049] Explanation of Table 1: "+" indicates that the detection line of the test strip develops color, and "-" indicates that the detection line of the test strip does not develop color.

[0050] Extraction of total DNA: Extract the total DNA of the above ten samples according to the method described in Blood / Cell / Tiss...

Embodiment 3

[0056] Embodiment 3: Sensitivity test of Babesia vole RPA detection method

[0057] The sensitivity of the RPA method for Babesia microti was tested using COX I gene-positive gDNA as a template. Methods as below:

[0058] 1. The method of embodiment 1 extracts blood total DNA template from BALB / c mouse blood sample.

[0059] 2. Amplify the target fragment by common PCR method: amplify the 18S rRNA gene of Babesia microti with the total blood DNA of infected BALB / c mice, and the nucleotide sequence of the primer pair is as follows.

[0060] Upstream primer F2: 5'-AACCTGGTTGATCCTGCCAGTAGTCAT-3';

[0061] Downstream primer R2: 5'-GATCCTTCTGCAGGTTCACCTAC-3';

[0062] The reaction system is: TaKaRa Taq enzyme (5U / μl) 0.3ul, 10×PCR Buffer (Mg 2+ Plus) 2.5ul, dNTPMixture (2.5mM each) ul, template DNA 2ul, ddH 2 O 16.2ul, 1ul each of upstream and downstream primers. The total reaction system is 25ul.

[0063] 3. PCR product analysis: 10 μl of the final PCR product above was add...

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Abstract

The invention discloses a field mouse babesia RPA molecular detection method. The field mouse babesia RPA molecular detection method comprises the following steps: designing a pair of specific primersand probes according to field mouse babesia mitochondria COX I gene sequence, wherein the nucleotide sequences of the primer pair is shown as SEQ ID NO: 2 to SEQ ID NO: 4 respectively; then extracting total DNA inside a dog blood sample to carry out recombinase combined enzymatic amplification reaction; and finally adopting a lateral flow analysis test paper strip to carry out detection on an amplification product. The method is high in sensitivity and high in specification, and field mouse babesia can be identified rapidly.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and in particular relates to a molecular detection method of recombinase polymerase isothermal amplification (Recombinase Polymerase Aplication, RPA) of Babesia microti. technical background [0002] Babesia vole is infected and spread through hard tick bites, blood transfusions, etc. It has no obvious seasonality and is widely distributed. The infected host will have severe clinical symptoms and even lead to death. source. Since the first human case was reported in Yugoslavia in 1957, human infection with Babesia microti has been reported in my country, but the pathogenic mechanism of this zoonotic disease is still unclear. [0003] Babesia vole can achieve vertical transmission in mice, which means that mice infected with the worm can use its strong reproductive ability to strengthen the spread of Babesia vole. Moreover, it can remain in the host for a long time by avoiding the ho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/6893C12Q1/04
CPCC12Q1/6844C12Q1/6893C12Q2531/119C12Q2521/507C12Q2521/101C12Q2565/625
Inventor 贺兰赵阳楠崔杰赵俊龙罗晓莹李慕晓安晓梦
Owner HUAZHONG AGRI UNIV
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