Bovine theileriosis detection kit

Abstract

The invention discloses a kit for bovine theileriosis isothermal amplification detection and an application method of the kit. The kit comprises a isothermal amplification primer, amplification reaction liquid and an immunochromatography test strip for result detection. The detection kit has the advantages that the kit is high in specificity and sensitivity; the amplification reaction is performedat constant temperature, the whole reaction process only needs 50 minutes, and result detection only needs 1-2 minutes; the kit does not rely on expensive instruments and is simple and quick to operate, low in detection cost and especially suitable for field fast detection of bovine theileriosis.

Description

technical field

[0001] The invention relates to a detection kit for animal parasites and a use method thereof, in particular to a detection kit for bovine theileriasis and a use method thereof for non-disease detection purposes. Background technique

[0002] Bovine theileriosis (Bovine theileriosis) is a general term for the diseases caused by the protozoa of the genus Theilerisis parasitic in the macrophages, lymphocytes, and erythrocytes of cattle transmitted by vector ticks. Sick cattle manifested as fever, anemia, jaundice, and enlarged lymph nodes on the body surface, and severe infection caused death. Currently reported Theileria infecting cattle include Theileria parva, Theileria annulata, Theileria orientalis, Theileria sinensis, Theileria mutans, Theileria annulata Theileria velifera et al. (Gubbels et al. 2000; Gubbels et al. 2002; Stewart et al. 1996; Uilenberg et al. 1985). In my country, there are three kinds of Theileria bovis reported: Theileria annuli, Thei...

Examples

Embodiment 1

[0023] Embodiment 1: the screening of optimal reaction temperature and reaction time

[0024] 1. Genomic DNA Extraction of Theileria annuli

[0025] Take 200 μL of bovine anticoagulant blood positive for Theileria annuli that was stored at -20°C, and use the QIAamp DNAMini Kit (Qiagen, Germany) to extract blood DNA. The specific operation method refers to the instruction manual.

[0026] 2. Primer Design

[0027] According to the 18S rRNA gene sequences of Theileria annulus, Theileria sinensis, Theileria orientalis, Babesia bovis, Babesia double buds, Babesia ovale, Babesia major, and Babesia orientalis published by GenBank, after comparative analysis , the cross-amplification primers used to amplify three kinds of Theileria were designed in the mutated segment, and the amplified length was about 170bp.

[0028] Replacement primer Bovis-T-5a: 5'-AGAAAATTAGAGTGCTCA-3' (SEQ ID No.1)

[0029] Replacement primer Bovis-T-4s: 5'-CAACTGTTCCTATTAACCAT-3' (SEQ ID No.2)

[0030] Cro...

Embodiment 2

[0045] Example 2: Specific detection of cross primer amplification

[0046] 1. Genomic DNA Extraction of Piroplasma bovis

[0047] Take Babesia bovis, Babesia double bud, Babesia major, Babesia ovale, Babesia orientalis, Theileria annulus, Theileria sinensis, Theileria orientalis, and Piroplasma-negative cattle that were stored at -20°C. Each 200 μL of anticoagulated blood was extracted with QIAamp DNA Mini-kit (Qiagen, Germany), and the specific operation steps were carried out according to the instructions.

[0048] 2. Isothermal amplification

[0049] Amplification system:

[0050]

[0051]

[0052] Place the reaction tube at 55°C for 50 minutes, and inactivate at 80°C for 2 minutes

[0053] 3. Amplification product detection

[0054] Take 10 μL of the above reaction product, drop it on the absorbent pad of the chromatography test strip, insert it into a 1.5ml centrifuge tube filled with 100 μL of chromatography buffer, let it stand for 3-5 minutes, and read the e...

Embodiment 3

[0060] Embodiment 3: sensitivity test

[0061] 1. Preparation of 18S rRNA recombinant plasmid standard products of Theileria annulus, Theileria sinensis and Theileria orientalis

[0062] The 18S rRNA recombinant plasmids of Theileria annulus, Theileria sinensis, and Theileria orientalis stored at -20°C were used to measure the plasmid concentration with a NanoDrop2000 (Thermo Scientific, America) ultramicro spectrophotometer, and convert it into copies / μL. Use this as a plasmid standard and dilute to 1.0×10 5 -1.0×10 0 copies / μL for sensitivity testing.

[0063] 2. Isothermal amplification

[0064] Amplification system

[0065]

[0066] The above-mentioned reaction tubes were reacted at 55°C for 50 minutes and inactivated at 80°C for 2 minutes.

[0067] 4. Analysis of amplification products

[0068] Take 10 μL of the above reaction product, add it dropwise to the absorbent pad of the chromatography test strip, insert it into a 1.5mL centrifuge tube filled with 100 μL ...

technical field

[0001] The invention relates to a detection kit for animal parasites and a use method thereof, in particular to a detection kit for bovine theileriasis and a use method thereof for non-disease detection purposes. Background technique

[0002] Bovine theileriosis (Bovine theileriosis) is a general term for the diseases caused by the protozoa of the genus Theilerisis parasitic in the macrophages, lymphocytes, and erythrocytes of cattle transmitted by vector ticks. Sick cattle manifested as fever, anemia, jaundice, and enlarged lymph nodes on the body surface, and severe infection caused death. Currently reported Theileria infecting cattle include Theileria parva, Theileria annulata, Theileria orientalis, Theileria sinensis, Theileria mutans, Theileria annulata Theileria velifera et al. (Gubbels et al. 2000; Gubbels et al. 2002; Stewart et al. 1996; Uilenberg et al. 1985). In my country, there are three kinds of Theileria bovis reported: Theileria annuli, Thei...

Examples

Embodiment 1

[0023] Embodiment 1: the screening of optimal reaction temperature and reaction time

[0024] 1. Genomic DNA Extraction of Theileria annuli

[0025] Take 200 μL of bovine anticoagulant blood positive for Theileria annuli that was stored at -20°C, and use the QIAamp DNAMini Kit (Qiagen, Germany) to extract blood DNA. The specific operation method refers to the instruction manual.

[0026] 2. Primer Design

[0027] According to the 18S rRNA gene sequences of Theileria annulus, Theileria sinensis, Theileria orientalis, Babesia bovis, Babesia double buds, Babesia ovale, Babesia major, and Babesia orientalis published by GenBank, after comparative analysis , the cross-amplification primers used to amplify three kinds of Theileria were designed in the mutated segment, and the amplified length was about 170bp.

[0028] Replacement primer Bovis-T-5a: 5'-AGAAAATTAGAGTGCTCA-3' (SEQ ID No.1)

[0029] Replacement primer Bovis-T-4s: 5'-CAACTGTTCCTATTAACCAT-3' (SEQ ID No.2)

[0030] Cro...

Embodiment 2

[0045] Example 2: Specific detection of cross primer amplification

[0046] 1. Genomic DNA Extraction of Piroplasma bovis

[0047] Take Babesia bovis, Babesia double bud, Babesia major, Babesia ovale, Babesia orientalis, Theileria annulus, Theileria sinensis, Theileria orientalis, and Piroplasma-negative cattle that were stored at -20°C. Each 200 μL of anticoagulated blood was extracted with QIAamp DNA Mini-kit (Qiagen, Germany), and the specific operation steps were carried out according to the instructions.

[0048] 2. Isothermal amplification

[0049] Amplification system:

[0050]

[0051]

[0052] Place the reaction tube at 55°C for 50 minutes, and inactivate at 80°C for 2 minutes

[0053] 3. Amplification product detection

[0054] Take 10 μL of the above reaction product, drop it on the absorbent pad of the chromatography test strip, insert it into a 1.5ml centrifuge tube filled with 100 μL of chromatography buffer, let it stand for 3-5 minutes, and read the e...

Embodiment 3

[0060] Embodiment 3: sensitivity test

[0061] 1. Preparation of 18S rRNA recombinant plasmid standard products of Theileria annulus, Theileria sinensis and Theileria orientalis

[0062] The 18S rRNA recombinant plasmids of Theileria annulus, Theileria sinensis, and Theileria orientalis stored at -20°C were used to measure the plasmid concentration with a NanoDrop2000 (Thermo Scientific, America) ultramicro spectrophotometer, and convert it into copies / μL. Use this as a plasmid standard and dilute to 1.0×10 5 -1.0×10 0 copies / μL for sensitivity testing.

[0063] 2. Isothermal amplification

[0064] Amplification system

[0065]

[0066] The above-mentioned reaction tubes were reacted at 55°C for 50 minutes and inactivated at 80°C for 2 minutes.

[0067] 4. Analysis of amplification products

[0068] Take 10 μL of the above reaction product, add it dropwise to the absorbent pad of the chromatography test strip, insert it into a 1.5mL centrifuge tube filled with 100 μL ...

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