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Screening of schistosoma japonicum W chromosome specific gene and application thereof in cercaria sex identification

A technology for specific genes and schistosomiasis, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of screening out nucleic acid sequences or genes, etc., to improve standardization and homogenization, and has a wide range of applications. highly reproducible results

Active Publication Date: 2019-08-20
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the genome of Schistosoma japonicum has not yet been assembled to the chromosome level, and it is impossible to screen out W chromosome-specific nucleic acid sequences or genes based on the genome sequence alone.
At present, there are few reports on molecular biology methods for sex identification of Schistosoma japonicum cercariae

Method used

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  • Screening of schistosoma japonicum W chromosome specific gene and application thereof in cercaria sex identification
  • Screening of schistosoma japonicum W chromosome specific gene and application thereof in cercaria sex identification
  • Screening of schistosoma japonicum W chromosome specific gene and application thereof in cercaria sex identification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Schistosoma japonicum W chromosome-specific gene screening

[0043] (1) 100 female-specific expression genes were preliminarily screened out based on the ratio of fluorescent signals expressed by male and female adults, and 12 candidate genes whose gene sequences were better compared to the Schistosoma japonicum genome were screened out through bioinformatics analysis.

[0044] (2) Design of PCR primers: According to the gene sequence, gene-specific primers were designed using PrimerPremier 5.0 software.

[0045] Table 1 Primer information of 12 female specific expression genes of Schistosoma japonicum

[0046]

[0047] (3) PCR screening of Schistosoma japonicum W chromosome-specific genes: the cDNA and genomic DNA of male and female adults of Schistosoma japonicum were respectively used as templates, the housekeeping gene PSMD4 was used as an internal reference gene, and the upstream primer was 5'-CCTCACCAACAATTTCCACATCT-3' (SEQ ID NO. 10), the downstream...

Embodiment 2

[0051] Example 2 Verification of Schistosoma japonicum W chromosome-specific genes in different geographical strains

[0052] (1) Genomic DNA extraction: collect the male and female adults of Schistosoma japonicum in Anhui, Hunan, Jiangxi and Jiangsu provinces in China, and use the Genomic DNA Extraction Kit (QIAGEN Company) to extract the genomic DNA of the male and female adults respectively (the female and male worms were biologically extracted three times respectively). duplicates) to prepare PCR templates.

[0053] (2) Using the genomic DNA of male and female adults of Schistosoma japonicum in different provinces as templates and PSMD4 as an internal reference gene, double-PCR detection was carried out using the primers of SjF4, SjF6 and SjF9 specific genes of Schistosoma japonicum W chromosome, respectively. The PCR reaction system is as follows:

[0054]

[0055] The PCR amplification procedure is the same as in Example 1.

[0056] (3) Detect the double PCR product...

Embodiment 3

[0057] Example 3 Preparation of positive snails infected by single-sex and mixed-sex Schistosoma japonicum

[0058] (1) Negative snail screening: Oncomelania were collected in non-schistosomiasis-endemic areas, and further verified as non-schistosomiasis-infected negative snails by the light escape method.

[0059] (2) Preparation of positive snails artificially infected: a single snail was infected by a single miracidia or a mixed infection of multiple miracidia was used to infect a snail, and the snails were cultured in a light incubator for 3 months after infection.

[0060] (3) Identification of cercariae sex by animal infection experiment: put one artificially infected snail into each test tube and number them, and release the cercariae in a single positive snail by light-escape method, and pick about 60 cercariae from each snail and pass them through the abdominal patch Mice were infected one-on-one, and the infected mice were fed for 5 weeks and then dissected to obtain...

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Abstract

The invention discloses screening of schistosoma japonicum W chromosome specific gene and an application thereof in cercaria sex identification, particularly relates to a double PCR method adopting aspecific primer containing the schistosoma japonicum W chromosome specific gene and a schistosoma japonicum reference gene PSMD4 primer, and belongs to the technical field of biology. The method usesthe schistosoma japonicum W chromosome specific gene primer and the PSMD4 house-keeping gene primer, and rapidly identifies the sex of schistosoma japonicum cercariae by using the double PCR method, and the whole process only needs 2 hours. Compared with traditional animal experiment identification methods, the method greatly shortens the identification time and improves the standardization and homogenization of a schistosomiasis japonica animal model. Meanwhile, the method does not need to extract cercaria DNA, is simple, convenient and rapid in operation and high in repeatability compared with the traditional PCR methods, and can realize high-throughput identification. The method not only can identify the single female or male cercariae released by positive oncomelania, but also can identify the male and female mixed-sex cercarias released by the positive oncomelania.

Description

technical field [0001] The invention relates to the field of biological technology, in particular to the screening of the W chromosome specific gene of Schistosoma japonicum and its application in the sex identification of cercariae. Background technique [0002] Schistosoma japonicum is a unique dioecious fluke. The chromosomes include 7 pairs of autosomes and 1 pair of sex chromosomes. The male sex chromosome is ZZ type, and the female sex chromosome is ZW type. Schistosoma japonicum, Schistosoma mansoni and Schistosoma haematobium are the three most important pathogens of human schistosomiasis. The unisexual females of Schistosoma japonicum cannot develop and mature in the host body, and their sexual maturation depends on encumbrance with males, and the large number of eggs produced by mature females is the key to causing pathological damage to the host and re-transmission of the disease. The development of rapid sex identification technology for schistosomiasis cercaria...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12Q1/6809C12N15/11
CPCC12Q1/6888C12Q1/686C12Q1/6809C12Q2600/124C12Q2600/16C12Q2600/158C12Q2521/537C12Q2537/143C12Q2531/113C12Q2563/107C12Q2565/125Y02A50/30
Inventor 刘帅陈启军侯楠朴贤玉
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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