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
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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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