High resolution melting real-time PCR detect and identify filarial parasites in domestic cats
Objective: To detect and identify filarial parasites in dried blood spots (DBS) collected from domestic cats using high resolution melting real-time PCR (HRM RT-PCR). Methods: A total of 208 DBS were collected from domestic cats in a brugian filariasis endemic areas in Surat Thani Province, southern...
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Wolters Kluwer Medknow Publications
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doaj-e9f605cb91f849f0a7aabb07b134ba0a2020-11-25T02:07:44ZengWolters Kluwer Medknow PublicationsAsian Pacific Journal of Tropical Medicine2352-41462018-01-01111268268710.4103/1995-7645.248340High resolution melting real-time PCR detect and identify filarial parasites in domestic catsDarawan NonsaithongSupit YotmekSomsri YotmekHathai NochoteSirichit WongkamchaiSittiruk RoytrakulUsa Lek-UthaiObjective: To detect and identify filarial parasites in dried blood spots (DBS) collected from domestic cats using high resolution melting real-time PCR (HRM RT-PCR). Methods: A total of 208 DBS were collected from domestic cats in a brugian filariasis endemic areas in Surat Thani Province, southern Thailand. Microfilariae were found in 9 blood slides using Giemsa-stained thick blood film. The extracted DNA from blood spot volumes of 10 and 20 μL DBS with positive filarial parasites in cats were performed using HRM RT-PCR method. The primers were designed based on the partial mitochondrial 12S rRNA gene for identifying Brugia malayi, Brugia pahangi, Dirofilaria immitis. All purified samples were then detected. Results: Using different volumes of 10 μL and 20 μL DBS could easily distinguish filarial parasites and showed similar results. PCR amplicons of Brugia malayi, Brugia pahangi and Dirofilaria immitis were determined at melting peak (temperature) of 75.70 77.46 and 73.56 respectively. All 9 positive DBS samples showed positive Brugia pahangi and similar nucleotide sequences. Conclusions: This HRM RT-PCR method is able to diagnose, identify and discriminate filarial parasites collected from DBS, which is simple and inexpensive compared with other probe-based genotyping methods. Furthermore, this method is useful to survey, prevent and control filariasis.http://www.apjtm.org/article.asp?issn=1995-7645;year=2018;volume=11;issue=12;spage=682;epage=687;aulast=Nonsaithonghigh resolution melting analysisdried blood spotbrugia malayibrugia pahangidirofilaria immitis |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Darawan Nonsaithong Supit Yotmek Somsri Yotmek Hathai Nochote Sirichit Wongkamchai Sittiruk Roytrakul Usa Lek-Uthai |
spellingShingle |
Darawan Nonsaithong Supit Yotmek Somsri Yotmek Hathai Nochote Sirichit Wongkamchai Sittiruk Roytrakul Usa Lek-Uthai High resolution melting real-time PCR detect and identify filarial parasites in domestic cats Asian Pacific Journal of Tropical Medicine high resolution melting analysis dried blood spot brugia malayi brugia pahangi dirofilaria immitis |
author_facet |
Darawan Nonsaithong Supit Yotmek Somsri Yotmek Hathai Nochote Sirichit Wongkamchai Sittiruk Roytrakul Usa Lek-Uthai |
author_sort |
Darawan Nonsaithong |
title |
High resolution melting real-time PCR detect and identify filarial parasites in domestic cats |
title_short |
High resolution melting real-time PCR detect and identify filarial parasites in domestic cats |
title_full |
High resolution melting real-time PCR detect and identify filarial parasites in domestic cats |
title_fullStr |
High resolution melting real-time PCR detect and identify filarial parasites in domestic cats |
title_full_unstemmed |
High resolution melting real-time PCR detect and identify filarial parasites in domestic cats |
title_sort |
high resolution melting real-time pcr detect and identify filarial parasites in domestic cats |
publisher |
Wolters Kluwer Medknow Publications |
series |
Asian Pacific Journal of Tropical Medicine |
issn |
2352-4146 |
publishDate |
2018-01-01 |
description |
Objective: To detect and identify filarial parasites in dried blood spots (DBS) collected from domestic cats using high resolution melting real-time PCR (HRM RT-PCR). Methods: A total of 208 DBS were collected from domestic cats in a brugian filariasis endemic areas in Surat Thani Province, southern Thailand. Microfilariae were found in 9 blood slides using Giemsa-stained thick blood film. The extracted DNA from blood spot volumes of 10 and 20 μL DBS with positive filarial parasites in cats were performed using HRM RT-PCR method. The primers were designed based on the partial mitochondrial 12S rRNA gene for identifying Brugia malayi, Brugia pahangi, Dirofilaria immitis. All purified samples were then detected. Results: Using different volumes of 10 μL and 20 μL DBS could easily distinguish filarial parasites and showed similar results. PCR amplicons of Brugia malayi, Brugia pahangi and Dirofilaria immitis were determined at melting peak (temperature) of 75.70 77.46 and 73.56 respectively. All 9 positive DBS samples showed positive Brugia pahangi and similar nucleotide sequences. Conclusions: This HRM RT-PCR method is able to diagnose, identify and discriminate filarial parasites collected from DBS, which is simple and inexpensive compared with other probe-based genotyping methods. Furthermore, this method is useful to survey, prevent and control filariasis. |
topic |
high resolution melting analysis dried blood spot brugia malayi brugia pahangi dirofilaria immitis |
url |
http://www.apjtm.org/article.asp?issn=1995-7645;year=2018;volume=11;issue=12;spage=682;epage=687;aulast=Nonsaithong |
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