96 well genomic dna extraction kit

From each sample, between 24 and 67 specimens were selected randomly and genomic DNA was isolated from the adductor muscles by using a 96‐well genomic DNA extraction kit according to the manufacturer's protocol (FAVORGEN Biotech Corp). DNA concentration was measured on a Nanodrop and diluted to 10 ng/μl. Two multiplex sets of four markers were used (set1: Med367, Med379, Med722 and Med733; set2: Med737, Med740, Med747 and Me15/16). Markers were used individually in the PCR (polymerase chain reaction), pooled per set and analysed on an ABI3730 DNA Analyser. Seven markers were microsatellite loci as described by Lallias, Stockdale, and Boudry (2009) and the Me15/16 locus targeted a part of the adhesive protein gene, which partially discriminates between M. edulis, M. galloprovincialis and M. trossulus (Inoue, Waite, Matsuoka, Odo, & Harayama, 1995). Marker information, PCR conditions and multiplex conditions are detailed in Table 2. The GeneScan 500 LIZ marker was used as an internal marker. Allele calling was performed in genemapper version 3.7 (Applied Biosystems 2004).

Genomic DNA was extracted from whole blood using the QIAamp 96 DNA blood kit (Qiagen, Valencia, CA) or the 96-well genomic DNA extraction kit (Favorgen, Taiwan) according to the manufacturer instructions. The presence of P. falciparum and P. vivax infections was determined by a post-PCR, ligation detection reaction—fluorescent microsphere assay (LDR-FMA: 2006 surveys) [30 (link)] and a qPCR assay (2010 survey) [31 (link), 32 (link)].

Whole blood samples, stored at −20 °C in microtainers were transferred on dry ice to the laboratory at the Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. DNA extraction from 200 to 300 μl of each blood sample was carried out using the Favorgen 96-Well Genomic DNA Extraction Kit, following the manufacturer’s recommendations. Malaria parasite infection was first identified by the QMAL assay, a genus-specific probe-based quantitative PCR (qPCR) targeting a conserved region of the 18S rRNA genes of Plasmodium [21 (link)], by using 4 μl of purified DNA equivalent to 8 μl of whole blood. For positive samples by QMAL, 4 μl DNA was subjected to species-specific 18S qPCR assays to detect P. vivax, P. ovale, and P. malariae as previously described [22 (link)]. For P. falciparum, the varATS qPCR assay was used [23 (link)]. Plasmodium knowlesi was detected with (forward primer: 5′-GTT AGC GAG AGC CAC AAA AAA GCG AAT-3′, reverse primer: 5′-ACT CAA AGT AAC AAA ATC TTC CAT A-3′, and hydrolysis probe: 5′-HEX-TGC TTT ATG TGC GCA TCC TCT ACC TA-BFQ-3′). All qPCR assays were performed with iTaq™ Universal Probe Supermix 2× on a CFX96 real-time PCR detection system.