Kit of dog echinococcus granulosus and echinococcus multilocularis based on POCKIT Micro fluorescent PCR platform, and application of kit

A technology of Echinococcus multilocularis and Echinococcus granulosus, applied in DNA/RNA fragments, microbe determination/inspection, biochemical equipment and methods, etc., which can solve problems such as inoperability, cross-reaction, and increased cost , to reduce testing costs and save time

Inactive Publication Date: 2017-11-21
SOUTHWEST UNIVERSITY FOR NATIONALITIES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the grass-roots animal quarantine department mainly uses ELISA method for the detection of echinococcosis in dogs and foxes, which has poor specificity and low sensitivity, and it is easy to cause cross-reaction with other parasites (such as Taenia polycephala, etc.), so it cannot be accurately determined
The specialized laboratory testing is mainly the PCR method, which requires a special fluorescent PCR instrument, which is expensive
In addition, these current PCR methods are all primers and probes designed separately for Echinococcus granulosus or Echinococcus granulosus. For one sample, it needs to be tested twice, which is time-consuming, increases costs, and may even be affected due to technical problems. Weak and inoperable

Method used

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  • Kit of dog echinococcus granulosus and echinococcus multilocularis based on POCKIT Micro fluorescent PCR platform, and application of kit
  • Kit of dog echinococcus granulosus and echinococcus multilocularis based on POCKIT Micro fluorescent PCR platform, and application of kit
  • Kit of dog echinococcus granulosus and echinococcus multilocularis based on POCKIT Micro fluorescent PCR platform, and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Kit based on POCKIT Micro fluorescent PCR canine granulosa and Echinococcus multilocularis

[0030] A test kit for detecting canine granulosus and Echinococcus multilocularis, comprising the following components:

[0031] (1) DNA template: DNA extracted from the specimen to be tested using the OMEGA Stool DNA Kit Genome Extraction Kit;

[0032] (2) Fluorescence quantitative PCR reaction solution: 3.5 μL (final concentration 600 nM) of upstream universal primers for granule and Echinococcus multilocularis, 3.5 μL (final concentration 600 nM) of downstream general primers for granule and Echinococcus multilocularis Echinococcus multilocularis universal probe 1.5 μL (final concentration 400 nM), Taq enzyme 1.5 μL (final concentration 5 U·μL -1 ), premixed 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;

[0033] The nucleotide sequence of the upstrea...

Embodiment 3

[0044] Example 3 Preparation and Sensitivity Detection of Echinococcus granulosus Standards Based on POCKIT Micro Fluorescent PCR Instrument

[0045] 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 80 copies / μl positive standard for sensitivity testing.

[0046] Select 3.5 μL of upstream and downstream primers for canine granulosus and Echinococcus multilocularis (final concentration 600 nM), 1.5 μL general probe for granulosus and Echinococcus multilocularis (final concentration 400 nM), 1.5 μL Taq enzyme (5 U·μL -1 ), premixed 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 Copy / 50μl Echinoc...

Embodiment 4

[0047] Example 4 Specific detection of Echinococcus granulosus based on POCKIT Micro fluorescent PCR instrument

[0048] According to the method of Example 2, the specific detection of Echinococcus granulosus was carried out, and the templates were Echinococcus granulosus G1 strain, Echinococcus multilocularis, Fasciola hepatica, Anterior and posterior disc flukes, Taenia polycephalum, Ascaris canis, and Giardia flagella Worm, Cryptosporidium, Taenia alveolar DNA, prepare 2 reaction systems for each worm body, respectively adopt POCKIT Micro of the present invention and ABI 7300 type PCR instrument to detect, wherein, what ABI 7300 type PCR instrument carries out is Real time PCR reaction, and the experimental results are shown in Table 2.

[0049] Table 2 Specific detection of Echinococcus granulosus based on POCKIT Micro fluorescent PCR instrument

[0050]

[0051] It can be seen from Table 2 that, compared with the detection method of ABI 7300 PCR instrument, this metho...

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Abstract

The invention discloses a kit of dog echinococcus granulosus and echinococcus multilocularis based on POCKIT Micro fluorescent PCR platform, and application of the kit. The kit comprises fluorescent quantitative PCR reaction fluid, positive comparison product and negative comparison product; the fluorescent quantitative PCR reaction fluid comprises upstream general primers of echinococcus granulosus and echinococcus multilocularis, downstream general primers of echinococcus granulosus and echinococcus multilocularis, general probes of echinococcus granulosus and echinococcus multilocularis, Taq enzyme; premixing buffer and deionized water. The nucleotide sequence of the upstream general primers of echinococcus granulosus and echinococcus multilocularis and the nucleotide sequence of the downstream general primers of echinococcus granulosus and echinococcus multilocularis are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. The kit design is specific to the general primers and the probes of two hydatids; the result can be determined by detection once, thus the time is greatly saved, and detection cost is reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit and application of canine granule and Echinococcus multilocularis based on the POCKIT Micro fluorescent PCR platform. Background technique [0002] Echinococcus granulosus and multilocular echinococcosis is a serious zoonotic parasitic disease, which is common in Sichuan, Qinghai, Gansu, Ningxia, Xinjiang and other regions in my country. Among them, yak and sheep are Echinococcus granulosa As the intermediate host of tapeworms, prairie rodents are the intermediate hosts of Echinococcus multilocularis, and both dogs and foxes are the final hosts of these two kinds of echinococcosis. On the one hand, cattle and sheep infected with Echinococcus granulosus seriously affect their growth performance, and on the other hand, the viscera of cattle and sheep infected with Echinococcus granulosus are swallowed by dogs and foxes without treatment, which is the key to causing the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2600/166C12Q2531/113C12Q2563/107
Inventor 郝力力汤承岳华黄建德
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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