Various important aquagenic zoonoses protozoa simultaneous assay kit and preparation method thereof

A technology for detection kits and water sources, which is used in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc. To solve problems such as cysts or small cysts, to achieve the effect of improving sensitivity, accurate and objective judgment, and strong specificity

Inactive Publication Date: 2011-08-17
JILIN UNIV
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  • Summary
  • Abstract
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AI Technical Summary

Problems solved by technology

[0005] The pathogens of the above three diseases are Giardia lamblia, Cryptosporidium parvum and Toxoplasma gondii, all of which are intracellular parasitic protozoa. Polluted water survives for a long time and is difficult to be killed by ordinary disinfectants, so it brings great difficulties to disease diagnosis, pathogen detection and environmental monitoring. It is urgent to develop a multiple rapid detection kit

Method used

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  • Various important aquagenic zoonoses protozoa simultaneous assay kit and preparation method thereof
  • Various important aquagenic zoonoses protozoa simultaneous assay kit and preparation method thereof
  • Various important aquagenic zoonoses protozoa simultaneous assay kit and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0057] 1. Design of primers and probes for multiplex rapid fluorescent quantitative PCR detection kit

[0058] The specific diagnostic genes of Giardia lamblia, Cryptosporidium parvum and Toxoplasma gondii were compared and selected in GeneBank as beta-giardin gene, TRAP-C2 gene and B1 gene, respectively.

[0059] According to the selected specific gene biological software Beacon Designer-v7.5, multiple fluorescent quantitative PCR primers and probes were designed as follows:

[0060] Giardia lamblia:

[0061] Ga: 5'-GGCATCTTCATTCATAGACCTTCTG-3'

[0062] Gs: 5'-ATATGCTGACATCTGGAGGAAAATC-3'

[0063] G-probe: 5'-FAM-TCTTCCGTCTCGCCGTAACTGGTAA-BHQ1-3'

[0064] Cryptosporidium parvum:

[0065] Ca: 5'-ATTGAAATAGATTCTGGTCCTTTGG-3'

[0066] Cs: 5'-CATATTCCCTGTCCCTTGAGTTG-3'

[0067] C-probe: 5'-JOE-CCGACCTCTCTCTCATTTGGTGACC-BHQ1-3'

[0068] Toxoplasma gondii:

[0069] Ta: 5'-ATTTGCCAGCGGGAAATACAG-3'

[0070] Ts: 5'-CCACAAGACGGCTGAAGAATG-3'

[0071] T-probe: 5'-Cy5-TTCTTGTG...

experiment example 1

[0088] Experimental example 1. Specificity experiment of multiplex fluorescent quantitative PCR detection system

[0089] The DNA of 10 negative control samples such as Eimeria, Cryptosporidium ansei, Trichinella, Trichomonas, Escherichia coli, Shigella, Staphylococcus aureus, Clostridium botulinum, Bacillus cholerae, and Salmonella was taken as Templates were detected with the established amplification system. see figure 2 .

[0090] The results showed that none of the control samples met the genus-specific detection criteria.

experiment example 2

[0092] Sensitivity experiment of multiplex fluorescent quantitative PCR detection system

[0093] The extracted total nucleic acids of Giardia lamblia, Cryptosporidium parvum and Toxoplasma gondii were quantified on a nucleic acid analyzer, and the concentrations were diluted to 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg. Multiple fluorescent quantitative PCR detection was carried out, and a negative control was set at the same time. The results show that the detection threshold of Giardia lamblia is 100fg, the detection threshold of Cryptosporidium parvum is 10fg, and the detection threshold of Toxoplasma gondii is 10fg, all of which belong to high sensitivity detection methods and meet the sensitivity detection standards, see image 3 .

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Abstract

The invention discloses a various important aquagenic zoonoses protozoa simultaneous assay kit, and further provides a preparation method of the kit. A primer and a probe are designed according to the special gene sequence of the giardia lamblia, the cryptosporidium parvum and the toxoplasma gondii by a multi-fluorescence quantitative PCR (polymerase chain reaction) specific assay method, the sensitivity is improved by optimizing the fluorescence quantitative PCR reaction system and condition, the amplification can be performed, and an amplification result can be directly and timely observed.The invention can be used for fast and exactly assaying three important aquagenic zoonoses protozoa, and has the characteristics of being precise in design, simple and easy to operate, high in sensitivity, high in specificity, exact and objective in judgment, and the like.

Description

technical field [0001] The invention discloses a simultaneous detection kit for multiple important water-borne zoonotic protozoa, which is used for rapid fluorescent quantitative PCR detection of Giardia lamblia, Cryptosporidium parvum and Toxoplasma gondii. The invention also provides the kit The preparation method of the kit belongs to the technical field of parasite detection. Background technique [0002] Giardiasis is an intestinal disease caused by Giardia, with diarrhea as the main symptom. One of the parasitic diseases. [0003] Cryptosporidiosis is a serious zoonosis caused by Cryptosporidium, which can cause severe diarrhea in mammals, especially ruminants and humans. At present, there are 27 species of Cryptosporidium that have been named, and there are many kinds of Cryptosporidium that can infect humans. Among them, the infection of Cryptosporidium parvum is the most common and serious. Cryptosporidium parvum has a wide range of hosts and serious harm. and ot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCY02A50/30
Inventor 张西臣李建华苏利波宫鹏涛王景林张国才杨举李赫李巍张楠任文陟
Owner JILIN UNIV
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