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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.
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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.
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OBJECTIVE@#To investigate the genetic polymorphism of Plasmodium vivax (P. vivax) PvCSP and PvMSP1 genes from field isolates at four endemic regions (North, East, West and South) of Thailand.@*METHODS@#The 152 P. vivax infected cases from dried blood spots were DNA extracted and confirmed by species-specific primer sets using multiplex PCR method. PvMSP1 fragments F2 and F3; PvCSP were genotyped using RFLP-PCR method.@*RESULTS@#Totally amplified DNA which was multiple genotypes for PvMSP1 F2 and PvMSP1 F3 were 12.50% and 8.55%, respectively while PvCSP was 3.95%. The overall frequency of multiple genotypes was 25%. There were 12 allele types of PvMSP1 F2 using AluI enzyme digestion and 8 size variations were found in PvMSP1 F3. The isolates from western region was highly genetic diverse when compare among all isolates. The predominant variant type of PvCSP gene was VK210 type.@*CONCLUSIONS@#The multiple genotypes are common found in Thailand and it might hide the real genotype. PvCSP does not have extensive genetic diversity in this study. However, PvMSP1 marker due to multiple genotypes is difficult to be analyzed. The multiple genotypes findings might stem from population migration and vector species findings.