Babesia motasi detection kit

A technology for describing Babesia mohei and a detection kit, which is applied to the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc. It is not conducive to problems such as rapid detection, and achieves the effect of simple operation, high sensitivity and high specificity

Inactive Publication Date: 2019-04-09
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The current method used to detect Babesia infection in sheep is the classic blood smear staining method. This method is difficult to detect the pathogen when the infection rate is very low, and requires high skills for operators.
Babesia identification and detection methods developed based on molecular biology include real-time fluorescent quantitative PCR, reverse linear blotting, multiplex PCR, etc. These methods have high sensitivity and high specificity, but real-time fluorescent quantitative PCR and multiplex PCR require expensive instruments ;Reverse linear blotting operation is cumbersome and time-consuming
And above-mentioned method is unfavorable for the application of on-the-spot rapid detection

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Optimization of optimal reaction temperature and time for constant temperature amplification of cross primers

[0045] 1. Blood Genomic DNA Extraction

[0046] Blood DNA was extracted with QIAamp DNAMini Kit (Qiagen, Germany), and the specific operation steps were carried out according to the instructions.

[0047] 2. Primer Design

[0048] According to the 18S rRNA gene sequences of Babesia mosoni, Xinjiang strain of Babesia undetermined species, Theileria ovis, Theileria yoshii, etc. published by GenBank, after comparative analysis, a cross-amplified sequence was designed for species conservation and interspecies variation. Amplifying primers, the target sequence selection is located at the 5' end of 18S rRNA, and the amplification length is 150bp.

[0049] Replacement primer LT-5a: 5'-GCTAATTGTAGGGCTAATACAAG-3'

[0050] Replacement primer LT-4s: 5'-CTTGAATGGAACATCGCTAA-3'

[0051] Cross primer LT-2a1s:

[0052] 5'-CGATGCCTTTTGGCGGCGCGATTCGCAAGTTTATTAT...

Embodiment 2

[0068] Example 2: Specific detection of cross primer amplification detection

[0069] 1. Genomic DNA extraction of Piroplasma sheep

[0070] The Lintan strain of Babesia molovi stored at -20°C was positive, the Babesia motsui Hebei strain was positive, the Babesia motsui Ningxian strain was positive, the Babesia undetermined species Xinjiang strain was positive, Theileria ovis was positive, Youshi 200 μL of sheep blood that was positive for Theileria and negative for Piroplasma was extracted with QIAamp DNA Mini Kit (Qiagen, Germany), and the specific operation steps were performed according to the instructions.

[0071] 2. Isothermal Amplification of Cross Primers

[0072] reaction system:

[0073]

[0074]

[0075] The above-mentioned reaction tubes were reacted at 63°C for 50 minutes and inactivated at 80°C for 2 minutes.

[0076] 3. Amplified product test strip detection

[0077] Take 10 μL of the above reaction product, drop it onto the absorbent pad of the chro...

Embodiment 3

[0083] Embodiment 3: the sensitivity test of cross primer amplification

[0084] 1. Babesia mosoni merozoite DNA extraction

[0085] Take 100 μL of purified merozoites of Babesia moschii Lintan strain stored at -80°C and extract them with QIAamp DNA Mini Kit (Qiagen, Germany). The specific operation steps refer to the instructions.

[0086] 2. Preparation of Genomic DNA Gradient Dilution Samples

[0087] The extracted genomic DNA samples of Babesia moseni merozoites were measured with a micro-nucleic acid analyzer (NanoDrop 2000, Thermo Scientific, USA). The concentration is 20ng / μL, and the genomic DNA sample is serially diluted with ultrapure water, 4ng / μL, 800pg / μL, 160pg / μL, 32pg / μL, 6.4pg / μL, 1.28pg / μL, 0.3pg / μL , 0.06pg / μL, 0.012pg / μL.

[0088] 3. Isothermal Amplification of Cross Primers

[0089] reaction system:

[0090]

[0091] The above-mentioned reaction tubes were reacted at 63°C for 50 minutes and inactivated at 80°C for 2 minutes.

[0092] 4. Analysis o...

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PUM

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Abstract

The invention discloses a kit for detecting and identifying babesia motasi through the combination of isothermal amplification of cross primers and immunochromatography and a use method of the kit. The babesia motasi detection kit comprises five primers for isothermal amplification, namely a cross primer with the sequence of SEQ ID No.3, two replacement primers with the sequences of SEQ ID No.1 and SEQ ID No.2, a probe primer with the sequence of SEQ ID No.4, and a biotin labelled probe primer with the sequence of SEQ ID No.5. The kit further comprises an immunochromatographic test strip. Thekit has the advantages of convenience in operation, speediness, high sensitivity, high specificity and wide scope of application, and the detection result can be read by naked eyes.

Description

technical field [0001] The present invention relates to a method and a kit for detecting and identifying whether animals carry pathogenic parasites for the purpose of non-disease diagnosis. Specifically, the present invention relates to a method and a kit for detecting and identifying pathogenic parasites by means of constant temperature amplification of cross primers combined with immunochromatography. Methods and kits for identifying Babesia moseni. Background technique [0002] Ovis Babesiosis in sheep is a blood protozoan disease transmitted by vector ticks and parasitic in the red blood cells of sheep and goats. The infected animals show fever, jaundice, and hemoglobinuria. death (Uilenberg, 2006). The pathogens causing sheep babesiosis reported worldwide include Babesia motasi, Babesia ovis, Babesia crassa, Babesia foliata and Babesia taylori. There are two species of Babesia prevalent in my country, namely, Babesia mosoni and the unspecified Xinjiang strain of Babe...

Claims

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Application Information

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IPC IPC(8): C12Q1/6804C12Q1/6844C12Q1/6893C12N15/11
CPCC12Q1/6804C12Q1/6844C12Q1/6893C12Q2565/625
Inventor 王锦明关贵全刘军龙刘志杰李有全殷宏罗建勋
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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