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Species-level identification of trypanosomes infecting Australian wildlife by High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR).

ABSTRACT: Conventional nested PCR and Sanger sequencing methods are currently the gold standards for detecting trypanosomes in wildlife. However, these techniques are time-consuming and can often overlook mixed infections. True trypanosome prevalence can thus be underrepresented. Here, we designed an 18S rDNA-based real-time quantitative PCR (qPCR) assay coupled with High-Resolution Melting Analysis (HRMA) to detect and discriminate three Trypanosoma species (T. copemani, T. noyesi, and T. vegrandis) commonly infecting Australian marsupials. A total of 68 genetically characterised samples from blood and tissue were used to validate the High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR) assay. A further 87 marsupial samples consisting of blood, tissue and in vitro cultures derived from wildlife blood samples, were screened for the first time using this assay, and species identity confirmed using conventional PCR and Sanger sequencing. All three Trypanosoma species were successfully detected in pure cultures using the HRM-qPCR assay, and in samples containing mixed trypanosome infections. Of the 87 marsupial samples screened using the HRM-qPCR assay, 93.1% were positive for trypanosomes, and 8.0% contained more than one trypanosome species. In addition to the three targeted Trypanosoma species, this assay was also able to detect and identify other native and exotic trypanosomes. The turnaround time for this assay, from sample preparation to obtaining results, was less than 2 h, with a detection limit of 10 copies of the amplicon in a reaction for each of the targeted trypanosome species. This more rapid and sensitive diagnostic tool provides a high throughput platform for the detection, identification and quantification of trypanosome infections. It will also improve understanding of host diversity and parasite relationships and facilitate conservation management decisions.

SUBMITTER: Keatley S

PROVIDER: S-EPMC7691731 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A rare viroid disease, Orange Spotting (OS), has been associated with Coconut cadang-cadang viroid variant (named OS-CCCVd) in oil palm. The low concentration of OS-CCCVd in oil palm leaf tissues makes it tedious to obtain high-quality RNA. Various conventional extraction protocols are available for extracting RNA; however, the bottleneck to the methods is the acquisition of good yields of high-quality RNA suitable for use in viroid detection. Studies looking into the automation of magnetic bead extraction systems for viroid detection in oil palm are limited. In this study, we have compared four extraction methods, namely the MagMAX™ mirVana Total RNA isolation kit (Mag-A), MagMAX™ plant RNA isolation kit (Mag-B), modification of MagMAX™ mirVana Total RNA isolation kit (Mag-Mod) and the convention method (NETME buffer). The KingFisher Flex System uses a 96-well plate format for the three automated approaches. The major modification in the Mag-Mod protocol is the inclusion of lithium chloride solution and NETME buffer to the lysis buffer (which enhances RNA recovery) and the reduction in the volume of reagents used per reaction and run conditions, including time. High-quality small RNA was produced as a result of altering the buffers and reagent volume in the Mag-Mod method, which also increased sample productivity (48 samples per day) and detection sensitivity. These effects indirectly decreased the number of technical replicates in a run. Importantly, the alteration permitted the use of ground plant tissue samples with a small sample amount of 0.05- 0.1 g as opposed to the 2-4 grequired with the conventional method. The development of the modified Mag-Mod method using the 96-deep well plate with a magnetic bead system vastly increased our sample throughput, complementing the detection methods (e.g. RT-PCR and HRM-qPCR). Aside from developing an improved extraction method (Mag-Mod method), a novel HRM-qPCR assay successfully differentiated OS-CCCVd variants and identified the presence of OS-CCCVd in samples, obviating the need for Sanger Sequencing.

Dataset Information

Species-level identification of trypanosomes infecting Australian wildlife by High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR).

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