POCKIT Micro fluorescent PCR platform-based cattle and sheep echinococcosis granulose detection kit, and applications thereof

A technology for echinococcosis and Echinococcus granulosus, which is applied in the field of detection kits for echinococcosis granulosus in cattle and sheep, can solve the problems of inability to accurately determine suspected cysts and the lack of grassroots quarantine departments, and achieve Time-saving and labor-saving reagents and consumables, saving reagents and consumables, the effect of simple preparation steps

Inactive Publication Date: 2017-06-20
SOUTHWEST UNIVERSITY FOR NATIONALITIES +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, whether the organs of cattle and sheep are infected with Echinococcus granulosus mainly relies on observation and touch, which has great limitations. For smaller cysts, or suspected cysts caused by other parasites (such as Fasciola hepatica, Cysticercus fine neck, etc.) cannot be accurately determined
The laboratory test is mainly based on the PCR method, which requires special instruments (PCR instrument, centrifuge, etc.), and the basic quarantine departments often lack corresponding instruments and technical personnel. The establishment of the method is imminent

Method used

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  • POCKIT Micro fluorescent PCR platform-based cattle and sheep echinococcosis granulose detection kit, and applications thereof
  • POCKIT Micro fluorescent PCR platform-based cattle and sheep echinococcosis granulose detection kit, and applications thereof
  • POCKIT Micro fluorescent PCR platform-based cattle and sheep echinococcosis granulose detection kit, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Kit for cattle and sheep echinococcosis based on POCKIT Micro fluorescent PCR

[0032] A kit for detecting echinococcosis in cattle and sheep, comprising the following components:

[0033] (1) DNA template: DNA extracted from the specimen to be tested using the PetNAD nucleic acid rapid extraction kit;

[0034] (2) Fluorescent quantitative PCR reaction solution: 3.5 μL of upstream primers for cattle and sheep echinococcosis (final concentration 400 nM), 3.5 μL of downstream primers for cattle and sheep echinococcosis (final concentration 400 nM), Coccidiosis probe 1.5 μL (final concentration 250 nM), Taq enzyme 1.5 μL (final concentration 5 U·μL -1 ), premix buffer 25 μL, deionized water 14 μL, the total amount is 49 μL, and the total amount of DNA template and fluorescent quantitative PCR reaction solution is 50 μL;

[0035] The nucleotide sequence of the upstream primer for cattle and sheep echinococcosis is shown in SEQ ID NO.1; the nucleotide sequence of...

Embodiment 3

[0046] Example 3 Preparation and Sensitivity Detection of Echinococcus granulosus Standards Based on POCKIT Micro Fluorescent PCR Instrument

[0047] Prepare the positive control substance according to the method of Example 1; adopt NanoDrop2000 to accurately quantify, then calculate the molar weight according to the molecular weight of the plasmid, and prepare 10 5 、10 4 、10 3 and 10 2 Positive standards in copies / μl were used for sensitivity testing.

[0048] Select 3.5 μL of upstream and downstream primers for echinococcosis granulosus (final concentration 400 nM), 1.5 μL probe for echinococcosis granulosus (final concentration 250 nM), 1.5 μL Taq enzyme (5 U·μL -1 ), premix buffer 25 μL, Echinococcus granulosus positive control template 1 μL (10 5 copy / 50μl), ddH 2 O 14 μL, a total of 50 μL, put into the PCR reaction tube, add an equal volume of 10 4 Copy / 50μl Echinococcus granulosus positive control template, 10 3 copies / 50 μl Echinococcus granulosus positive contr...

Embodiment 4

[0049] Example 4 Specific detection of Echinococcus granulosus based on POCKIT Micro fluorescent PCR instrument

[0050] According to the method of Example 2, the specific detection of Echinococcus granulosus was carried out, and the templates were the DNA of Echinococcus granulosus G1 strain, Echinococcus multilocularis, Anterior and posterior disc flukes, Fasciola hepatica, Taenia alveolar and Schistosoma japonicum , two reaction systems were prepared for each worm, and the POCKIT Micro and ABI 7300 PCR instruments of the present invention were used for detection respectively. Wherein, the ABI 7300 PCR instrument carried out the Real time PCR reaction, and the experimental results are shown in Table 2.

[0051] Table 2 Specific detection of Echinococcus granulosus based on POCKIT Micro fluorescent PCR instrument

[0052]

[0053] It can be seen from Table 2 that, compared with the detection method of ABI 7300 PCR instrument, this method has strong specificity, and can onl...

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Abstract

The invention discloses a POCKIT Micro fluorescent PCR platform-based cattle and sheep echinococcosis granulose detection kit, and applications thereof. The POCKIT Micro fluorescent PCR platform-based cattle and sheep echinococcosis granulose detection kit comprises a DNA extracting solution, a fluorogenic quantitative PCR reaction liquid, a positive reference substance, and a negative reference substance; the fluorogenic quantitative PCR reaction liquid comprises a cattle and sheep echinococcosis granulose upstream primer, a cattle and sheep echinococcosis granulose downstream primer, a cattle and sheep echinococcosis granulose probe, Taq enzyme, premixed buffer, and deionized water. The nucleotide sequences of the cattle and sheep echinococcosis granulose upstream primer, the cattle and sheep echinococcosis granulose downstream primer, and the cattle and sheep echinococcosis granulose probe are represented by SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 respectively. The POCKIT Micro fluorescent PCR platform-based cattle and sheep echinococcosis granulose detection kit is high in specificity and sensitivity; no pollution is caused; electrophoresis is not necessary; and one-site rapid detection can be realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection kit for echinococcosis of cattle and sheep based on the POCKIT Micro fluorescent PCR platform and its application. Background technique [0002] Echinococcus granulosus cercarosis is a serious zoonotic parasitic disease, which is widespread in Sichuan, Qinghai, Gansu, Ningxia, Xinjiang and other regions in my country, among which yaks and sheep are the main intermediate hosts. On the one hand, the growth performance of sheep infected with Echinococcus granulosus is seriously affected. On the other hand, the viscera of cattle and sheep infected with Echinococcus granulosus is swallowed by dogs without treatment, which is the key link causing the epidemic. [0003] At present, whether the organs of cattle and sheep are infected with Echinococcus granulosus mainly relies on observation and touch, which has great limitations. For smaller cysts, or suspected cysts c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/158C12Q2563/107C12Q2545/114C12Q2545/113
Inventor 郝力力汤承岳华黄建德
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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