Rapid and high throughput molecular identification of diverse mosquito species by high resolution melting analysis

Mosquitoes are a diverse group of invertebrates, with members that are among the most important vectors of diseases. The correct identification of mosquitoes is paramount to the control of the diseases that they transmit. However, morphological techniques depend on the quality of the specimen and often unavailable taxonomic expertise, which may still not be able to distinguish mosquitoes among species complexes (sibling and cryptic species). High resolution melting (HRM) analyses, a closed-tube, post-polymerase chain reaction (PCR) method used to identify variations in nucleic acid sequences, has been used to differentiate species within the Anopheles gambiae and Culex pipiens complexes. We validated the use of PCR-HRM analyses to differentiate species within Anopheles and within each of six genera of culicine mosquitoes, comparing primers targeting cytochrome b (cyt b), NADH dehydrogenase subunit 1 (ND1), intergenic spacer region (IGS) and cytochrome c oxidase subunit 1 (COI) gene regions. HRM analyses of amplicons from all the six primer pairs successfully differentiated two or more mosquito species within one or more genera (Aedes (Ae. vittatus from Ae. metallicus), Culex (Cx. tenagius from Cx. antennatus, Cx. neavei from Cx. duttoni, cryptic Cx. pipiens species), Anopheles (An. gambiae s.s. from An. arabiensis) and Mansonia (Ma. africana from Ma. uniformis)) based on their HRM profiles. However, PCR-HRM could not distinguish between species within Aedeomyia (Ad. africana and Ad. furfurea), Mimomyia (Mi. hispida and Mi. splendens) and Coquillettidia (Cq. aurites, Cq. chrysosoma, Cq. fuscopennata, Cq. metallica, Cq. microannulatus, Cq. pseudoconopas and Cq. versicolor) genera using any of the primers. The IGS and COI barcode region primers gave the best and most definitive separation of mosquito species among anopheline and culicine mosquito genera, respectively, while the other markers may serve to confirm identifications of closely related sub-species. This approach can be employed for rapid identification of mosquitoes.

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Rapid identification of Listeria species and screening for variants by melting curve and high-resolution melting curve analyses of the intergenic spacer region of the rRNA gene

Canadian Journal of Microbiology ◽

10.1139/w10-054 ◽

2010 ◽

Vol 56 (8) ◽

pp. 676-682 ◽

Cited By ~ 13

Author(s):

Jun Wang ◽

Shoichi Yamada ◽

Eiji Ohashi

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

Melting Curve ◽

Intergenic Spacer ◽

Rapid Identification ◽

Curve Analysis ◽

Melting Curve Analysis ◽

Rrna Gene ◽

Intergenic Spacer Region ◽

Listeria Species

The presence of any Listeria species in food may be an indicator of poor hygiene in food processing facilities. The biochemical identification of Listeria species is laborious and time consuming. Therefore, the development of novel identification methods that are rapid and simple to perform would be an asset. In this study, large intergenic spacer region amplicons of 343–374 bp were generated from 207 Listeria isolates. The melting curve analysis of these amplicons specifically classified all isolates into 6 Listeria species and generated 11 high-resolution melting (HRM) curve profiles. In this study, 3 HRM profiles were found in Listeria monocytogenes and Listeria innocua , and 2 were found in Listeria seeligeri . Sequencing of the amplicons representing these profiles revealed that each profile related to a unique sequence. The smallest difference recognized in this study was 1 nt. The results represented in this study show that HRM curve analysis of Listeria intergenic spacer sequences is a simple, quick, and reproducible method of simultaneously identifying 6 Listeria species and screening for variants. In particular, the completion of both reaction and analysis in a closed tube saves time by eliminating the separate steps and lowers the risk of contamination.

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