Primer group and kit for detecting diarrhea-causing parasites through multi-PCR technology

A parasite and primer set technology, applied in the biological field, can solve the problems of high missed detection rate, difficulty in comprehensive screening of diarrhea-causing parasite detection technology, etc., and achieve improved sensitivity, high specificity, high conservation and specificity Effect

Active Publication Date: 2017-02-15
北京卓诚惠生生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to solve the problems of difficulty in comprehensive screening and high missed detection rate in the existing detection technology of diarrhea-causing parasites, and to provide a new detection primer and method for diarrhea-causing parasites

Method used

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  • Primer group and kit for detecting diarrhea-causing parasites through multi-PCR technology
  • Primer group and kit for detecting diarrhea-causing parasites through multi-PCR technology
  • Primer group and kit for detecting diarrhea-causing parasites through multi-PCR technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] 1. Primer synthesis: the primers of SEQ ID NO.1 and 3-20 were synthesized. Taking the amount of substance as the unit, take 1 part of each of the primers of SEQ ID NO.3-20 and mix with 4 parts of the primer of SEQ ID NO.1 to form the primer set of this embodiment. See Table 3 for a summary of primers.

[0088] Table 3 Summary of Multiplex PCR Primer Sequences

[0089]

[0090]

[0091] 2. Specificity verification:

[0092] Select 12 unrelated pathogens as simulated interference samples: Salmonella (purchased from China Medical Culture Collection Center, No. 50001), Shigella (purchased from China Medical Culture Collection Center, No. 51054), Vibrio parahaemolyticus (purchased from China Medical Culture Collection Center, No. 21617), Campylobacter jejuni (purchased from China Medical Culture Collection Center, 186089), Staphylococcus aureus (purchased from China Medical Culture Collection Center, No. 26003), wax Bacillus-like bacteria (purchased from China Medic...

Embodiment 2

[0101] 1. Construction of multiplex PCR detection kit

[0102] The kit consists of 2× reaction system buffer, DNA polymerase, 10× primer mixture, positive control, and ultrapure water. The specific components are as follows: 2×PCR Buffer (Tris-HCl 40Mm (pH 8.3), KCl 100mM, tween-200.08%, 0.0006ng / μL PET28a, 1mM dNTP, 8mM MgCl 2 ); 25× DNA polymerase (2U / μL); 10× primer mixture (concentration of long primer pair including IAC is 2 μM each, concentration of homologous universal primer SEQ ID NO.1 is 8 μm); positive control (containing 8 mixed templates of parasites, each at 0.2 ng / μL).

[0103] 2. Genome extraction

[0104] Entamoeba histolytica, Giardia, Cryptosporidium, Schistosoma japonicum, Clonorchis sinensis, Strongyloides sternum, Blastocystis hominis and Paragonimus wescheri were selected as the target parasites for the establishment of the detection method, respectively The eight kinds of parasites were collected according to the national standard or industry standar...

Embodiment 3

[0114] The minimum detection limit test of embodiment 3 kit

[0115] Test samples for evaluation: select 8 parasites, sample 1 (template 1) is Entamoeba histolytica, sample 2 (template 2) is Giardia, sample 3 (template 3) is Cryptosporidium, sample 4 (Template 4) is Schistosoma japonicum, Sample 5 (Template 5) is Clonorchis sinensis, Sample 6 (Template 6) is Strongyloides sternum, Sample 7 (Template 7) is Blastocystis hominis, and Sample 8 (Template 8) is Paragonimus westermani, sample 9 (template 9) is a mixed template of the above eight parasites. The bacterial suspensions of the 9 templates were adjusted to 20ng / ul respectively, and the 9 samples were serially diluted into 2ng / ul, 0.2ng / ul, 0.02ng / ul, 0.002ng / ul and 0.0002ng / ul test samples.

[0116] Templates with different dilutions were detected using the kit of the present invention. Carry out operation and result judgment according to embodiment 2, the minimum detection limit test result that kit detects 8 target gen...

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Abstract

The invention provides a primer group for detecting diarrhea-causing parasite genes through the multi-PCR technology. The primer group comprises primers shown as SEQ ID NO.1 and SEQ ID 3-18. The invention further provides a detection method and a kit for detecting the diarrhea-causing parasite genes through the multi-PCR technology. After the technical scheme is adopted, a complete technical means is provided for the rapid and accurate screening of the diarrhea-causing parasite genes, the rapid screening and identification for the diarrhea-causing parasites after the food poisoning event are realized, and a reliable basis is provided for reasonably and properly disposing the diseases.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer set and a kit for multiplex PCR detection of diarrhea-causing parasites and a detection method thereof. Background technique [0002] Infectious diarrhea is a group of diseases with the highest incidence, the widest prevalence and serious impact among infectious diseases in my country. At present, the research on diarrhea caused by parasitic infection is relatively weak. Diarrhea caused by parasites has acute diarrhea and chronic diarrhea clinically, but most of them are chronic diarrhea, or often recur after an acute attack. [0003] There are many types of parasites that can cause diarrhea in my country, with wide distribution, high infection rate, great harm, and different prevalence in different regions. At present, in the diagnosis of parasitic diseases, etiological examination is the main basis for the diagnosis of parasitic infections. The clinical diagnosis is sti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/16C12Q2537/143Y02A50/30
Inventor 王雷王彦威王晓艳张志强
Owner 北京卓诚惠生生物科技股份有限公司
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