2021 Fiscal Year Research-status Report
Elucidating transmission dynamics for soil-transmitted helminths based on environmental DNA analysis
| Project/Area Number |
21K12269
|
|---|
| Research Institution | Dokkyo Medical University |
|---|
Principal Investigator |
オオタケサトウ マルセロ 獨協医科大学, 医学部, 助教 (50771896)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
迫 康仁 旭川医科大学, 医学部, 教授 (40312459)
サトウ 恵 新潟大学, 医歯学系, 准教授 (70601813)
|
|---|
| Project Period (FY) |
2021-04-01 – 2024-03-31
|
| Keywords | Ecohealth / One-health / environmental DNA / STH / qPCR |
|---|
| Outline of Annual Research Achievements |
Goal: Development of soil transmitted helminths-specific DNA detection method.
The DNA sequences of the parasite species causing STH were retrieved from the database, aligned and analyzed. We could obtain sequences from the following species: Ascaris lumbricoides, Trichuris trichiura and Hookworms (Ancylostoma duodenale, Ancylostoma ceylanicum, Necator americanus). Moreover, other species DNA sequences were also retrieved, aligned, and analysed once they are also found in STH endemic areas, namely: Ascaris suum, Toxocara canis, Toxocara cati, Ancylostoma caninum, Ancylostoma braziliense.
To design the environmental DNA detection assay for soil samples, we choose mitochondrial and nuclear genes. The target genes were mitochondrial DNA 16S rRNA, COX1 gene, or nuclear DNA ITS1, ITS2, which are often used to distinguish parasite species. From the aligned sequences of each gene by species of parasite we could determine intraspecific and interspecific variable and conserved areas which allowed us to design species specific sets of primers and probes with an expected short length of the amplified DNA (about 100 to 200 bp). All the obtained species-specific assays were screened in silico for specificity. At least 3 sets of primers-probe for each STH species were chosen to proceed to sensitivity and interspecies specificity tests.
|
| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
The project is progressing smoothly at the laboratory, however field samples are still not available.
Thus, we are concerned about the postponed field study due to restrictions for travelling to Thailand once it was expected to collect field samples for use to test sensitivity and specificity of our newly developed STH detection assays.
|
| Strategy for Future Research Activity |
1)We expect to resume postponed field studies in collaboration with Mahidol University.
2)Regarding the developing STH detection tests, using the best (in silico) species-specific assays (primers-probe set) for each STH species:
-we will evaluate the sensitivity of the developed soil-borne helminth-specific DNA detection method using samples with confirmed DNA concentrations (1 to 10000 copies). We expect to set the detection limit to about 20 copies.
-we will also conduct specificity tests using parasite samples different from the target species to be detected, and check whether each primer and probe show cross-reactivity. If there is a problem with specificity or sensitivity at this stage, improve of the DNA detection method will be needed.
|
| Causes of Carryover |
The reason for the incurring amount to be used next fiscal year is basically the travel restrictions due the COVID19 pandemics.
We hope to use the expected amount for the postponed field surveys and attend international meetings.
|
