Methods in Anopheles Research - MR4

Chapter 8 : Field Techniques8.1 Molecular Identification of Plasmodium spp. in AnophelinesPage 2 of 4Semi-nested multiplex PCR for Human Plasmodium species identification (Lardeux et al. 2008)Prepare PCR Master Mix for 96, 48 or 1 20 μl PCR reactions 1 . Add reagents in the order presented.96 48 1 Reagent259.2 µl 129.6 µl 2.7 µl Nuclease free H 2 O384.0 µl 192.0 µl 4.0 µl Taq 1X PCR Buffer with MgCl 296.0 µl 48.0 µl 1.0 µl dNTP’s (0.5 mM mix)96.0 µl 48.0 µl 1.0 µl Universal reverse. UNR (R, 0.05 µM) GAC GGT ATC TGA TCG TCT TC96.0 µl 48.0 µl 1.0 µl Plasmodium. PLF (F, 0.05 µM) AGT GTG TAT CAA TCG AGT TTC28.8 µl 14.4 µl 0.3 µl Go Taq DNA Polymerase ( 5U/µl)960 µl 480 µl 10 µl Total (to each 10µl reaction add 10 µl template DNA)Table 8.1.2. F and R indicate forward and reverse orientation.Prepare PCR 2 Master Mix for 96, 48 or 1, 20 µl PCR reactions; Add reagents in the order presented96 48 1 Reagent912.0 µl 456.0 µl 9.5 µl Nuclease free H 2 O384.0 µl 192.0 µl 4.0 µl Taq 1X PCR Buffer with MgCl 296.0 µl 48.0 µl 1.0 µl dNTP’s (0.5 mM mix)153.6 µl 76.8 µl 1.6 µl PLF (F, 0.08 µM) AGT GTG TAT CAA TCG AGT TTC38.4 µl 19.2 µl 0.4 µl P. falciparum. FAR (R, 0.04 µM) AGT TCC CCT AGA ATA GTT ACA38.4 µl 19.2 µl 0.4 µl P. vivax. VIR (R, 0.04 µM) AGG ACT TCC AAG CCG AAG C38.4 µl 19.2 µl 0.4 µl P. malariae. MAR (R, 0.04 µM) GCC CTC CAA TTG CCT TCT G38.4 µl 19.2 µl 0.4 µl P. ovale. OVR (R, 0.04 µM) GCA TAA GGA ATG CAA AGA ACA G28.8 µl 14.4 µl 0.3 µl Go Taq DNA Polymerase ( 5U/µl)1.73 ml 864 µl 18 µl Total (to each 18 µl reaction add 2 µl of PCR1 amplicon)Table 8.1.3. F and R indicate forward and reverse orientation.PCR cycle conditionsPCR 1 : UNR-PLF94°C/ 5 min x 1 cycle(94°C/ 1 min, 60°C/ 1 min, 72°C/ 90 sec) x 40 cycles4°C holdPCR 2: PLF-MAR, FAR, VIR, OVR94°C/ 5 min x 1 cycle(94°C/ 30 sec, 62°C/ 30 sec, 72°C/ 60 sec) x 35 cycles72°C/ 10 min x 1 cycle4°C holdRun samples on a 1.5 % agarose EtBr gel for visualization.If needed, positive samples from agarose can also be run on an 8% polyacrylamide gel, staining with0.2% silver nitrate and revealed with a 2:1 volume of 30 g / l sodium carbonate: 0.02% formaldehyde.Primers create fragments of 269 bp (P. malariae) , 395 bp (P. falciparum), 436 bp (P. ovale), 499 bp (P.vivax)1 Amounts for larger master mixes have been adjusted upwards to be sufficient for 50 and 100 rxnscompensate for imprecise measurements.