Liver echinococcus gene segment screening method, amplification primer and kit

A technology of gene fragments and screening methods, applied in biochemical equipment and methods, DNA/RNA fragments, bioinformatics, etc., can solve the problems of not using the whole genome of tapeworms, prone to false positive results, poor specificity, etc., and achieve enhanced Clinical application effect, high accuracy and strong specificity

Pending Publication Date: 2021-09-07
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Similarly, this diagnostic method also uses the multiplex PCR method, and the design of the primers is based on the mitochondrial gene reference sequences of Echinococcus granulosus in the narrow sense, Echinococcus canadensis and Echinococcus multilocularis. Excluding the influence of human genes and other tapeworm genes, false positive results are prone to occur in clinical application, and the specificity is poor or even unable to amplify

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  • Liver echinococcus gene segment screening method, amplification primer and kit
  • Liver echinococcus gene segment screening method, amplification primer and kit
  • Liver echinococcus gene segment screening method, amplification primer and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Such as figure 1 The method for screening gene fragments of liver echinococcosis comprises the following steps:

[0060] Obtain genome data, download the genome data of Echinococcus granulosus, Echinococcus multilocularis, close relatives, and human beings in the Genbank database, and the genome data is the whole genome sequence;

[0061] Excluding the influence of human genes, the genome of Echinococcus granulosus and the genome of Echinococcus multilocularis were compared with the human genome, and the same gene fragments in the genome of Echinococcus granulosus as the human genome were excluded, and the first Echinococcus granulosus was obtained. Coccidioides gene fragments; exclude the same gene fragments in the genome of Echinococcus multilocularis as the human genome, and obtain the first Echinococcus multilocularis gene fragments; use the supercomputing platform for comparison and calculation, and compare the genome of Echinococcus granulosus Carry out BLAST com...

Embodiment 2

[0072] The amplification primers designed based on the gene fragments screened by the screening method in Example 1, the amplification primers include at least one of the first amplification primer, the second amplification primer and the third amplification primer, wherein, The first amplification primer is used to amplify the third Echinococcus granulosus gene fragment, the second amplification primer is used to amplify the third Echinococcus multilocularis gene fragment, and the third amplification primer is used to amplify the common Gene fragment.

[0073] In this embodiment, the amplification primers include at least one of a first amplification primer, a second amplification primer, and a third amplification primer, wherein the first amplification primer is used to amplify the third cell The Echinococcus granulosus gene fragment, the second amplification primer is used to amplify the third Echinococcus multilocularis gene fragment, and the third amplification primer is ...

Embodiment 3

[0077] The amplification primers in Example 2 were further screened to determine 3 pairs of primer pairs that can accurately detect echinococcosis in human tissues. The primer pairs include Echinococcus granulosus-specific primer pairs and Echinococcus multilocularis-specific primer pairs. At least one of the pair of primers, and a pair of common detection primers, wherein,

[0078] Echinococcus granulosus-specific primer pairs:

[0079] Upstream primer: 5'-AAGCCGCCCTATTTAAAGTAGGAG-3' (SEQ ID NO.1)

[0080] Downstream primer: 5'-GAGAGATTTTCAAGCATTACGTTATCTGT-3' (SEQ ID NO.2)

[0081] Echinococcus multilocularis-specific primer pairs:

[0082] Upstream primer: 5'-GATGACTGGCTGACTGTAAGTAA-3' (SEQ ID NO.3)

[0083] Downstream primer: 5'-TTGAATAGCCGTAGGATAGTAAGAG-3' (SEQ ID NO.4)

[0084] Common Detection Primer Pairs:

[0085] Upstream primer: 5'-TGGCGAGATCTACCTGGGAA-3' (SEQ ID NO.5)

[0086] Downstream primer: 5'-CGTAAATGCAATCATGTCCGATCTAA-3' (SEQ ID NO.6)

[0087] Among t...

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Abstract

The invention discloses a liver echinococcus gene segment screening method, an amplification primer and a kit.The screening method comprises the following steps: eliminating an influence of a human genome and a close genetic relationship tapeworm group genome from whole genomes of echinococcus granulosus and echinococcus multilocularis; and screening to obtain a third echinococcus granulosus gene segment, a third echinococcus multilocularis gene segment and a common gene segment, and designing three types of amplification primers by using three types of the gene segments respectively. A primer pair group for detecting echinococcosis of human tissues is obtained by further screening and a kit and a use method of the kit are provided based on the primer pair group. False positive results caused by human genes or close genetic relationship tapeworm genes existing in to-be-detected tissue DNA is avoided from the source, the to-be-detected DNA aiming at the primer during design is a human tissue sample, the false negative results in clinical detection are remarkably reduced, specific primers have higher accuracy and higher specificity, and clinical use effects of the primer pair and the kit are obviously enhanced.

Description

technical field [0001] The invention relates to the field of echinococcosis diagnosis, in particular to a method for screening liver echinococcosis gene fragments, primers screened based on the method, primers, a kit and a detection method for detecting echinococcosis in human tissues. Background technique [0002] Hydatid disease, also known as echinococcosis, is a parasitic disease caused by echinococcus larvae parasitizing in humans or animals. my country's Tibet, Xinjiang, Ningxia, Gansu, Inner Mongolia, Qinghai and other areas with developed animal husbandry are endemic areas of echinococcosis, and the threatened population is close to 66 million. Tibet is one of the provinces with the worst echinococcosis epidemic in the country. Surveys show that the average prevalence of echinococcosis in endemic areas is 1.66%, about 49,900 people, and the number of early-stage infections is no less than 100,000. [0003] The infection of echinococcosis can cause damage to organs a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11G16B30/10
CPCC12Q1/6888C12Q1/686G16B30/10C12Q2565/125C12Q2547/101
Inventor 曾勇吴泓廖明恒袁克非徐琳
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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