Background
Methods
Plasmodium species reference strains and Plasmodium infected Anopheles
Mosquito preparation and dissection
DNA extraction using a cetyltrimethylammonium bromide (CTAB)-based method
18s-rRNA nested PCR
Diagnostic description | Reagents quantities and final concentration | Thermal profile |
---|---|---|
18s-rRNA genus specific PCR nest-1 [19] | 1X PCR buffer, 80 μM dNTPmix, 0.8 mM MgCl2, 0.1 mM each primer (rPLU1–rPLU5), 0.25 U Taq polymerase | 94 °C for 4 min; 35 cycles of 94 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min; and 72 °C for 4 min. Time: 120 min |
18s-rRNA genus specific PCR nest-2 [19] | Same as nest-1 but using rPLU3–rPLU4 primers | Same as nest-1 but annealing temperature is 62 °C. Time: 120 min |
Conventional COX-I PCR | 1X PCR buffera, 10 mM dNTPs, 0.4 mM each primer, 1.5 mM MgCl2 and 0.2 µL of recombinant Taq polymerase | 94 °C for 5 min; 40 cycles of 94 °C for 1 min, 62 °C for 1 min, 72 °C for 90 s; and 72 °C for 10 min. Time: 155 min |
Fast COX-I PCR | 1X blood phusion buffera, 1 mM each primer, and 0.125 µL of blood phusion polymerase | 98 °C for 4 min; 70 cycles of 98 °C for 1 s, 69 °C for 5 s, 72 °C for 35 s; and 72 °C for 10 min. Time: 62 min |
Single step PCR for Plasmodium sporozoite detection based on the cytochrome oxidase I
Fast COX-I PCR sequencing reaction
Results
Comparison of COX-I PCRs and 18s-rRNA nested-PCR using DNA (serial dilutions) of Plasmodium reference strains
Sample | Species IDa
| E valueb
| Coveragec (%) | Identityd
| [GenBank identifier]e
|
---|---|---|---|---|---|
PCR product from dilution 6 (0.043 pg of DNA) | |||||
P. falciparum
|
P. falciparum
| 0 | 100 | 99.7% | [KM065500.1] |
P. vivax
|
P. vivax
| 1.3e–84 | 100 | 98.4% | [KF668441.1] |
P. knowlesi
|
P. knowlesi
| 8e–180 | 100 | 100% | [AB444108.1] |
Known infective mosquitoes from the field (Kenya Highlands) | |||||
Mosquitoes (n = 21) |
P. falciparum
| 0 | 100 | 99.7% | [KM065500.1] |
Mosquitoes (n = 3) |
P. ovale s.l.
| 1.21e–151 | 100 | 97.5% | [KF018660.1] |
Positive mosquitoes from Western Province, Solomon Islands | |||||
Mosquitoes (n = 17) |
P. falciparum
| 0 | 100 | 99.7% | [KM065500.1] |
Mosquitoes (n = 5) |
P. vivax
| 0 | 99 | 99.6 | [KF668441.1] |
Mosquito (n = 1) |
P. ovale wallikeri
| 7.1e–110 | 100 | 92.2% | [HQ712053.1] |
Sensitivity and specificity between 18s-rRNA nested PCR and fast COX-I PCR for Plasmodium detection
Presence of Plasmodium infective mosquitoes from the Solomon Islands
Discussion
Molecular sporozoite detection approach [ref] | DNA extraction |
Plasmodium species | Cycling time in min | DNA limit of detection |
---|---|---|---|---|
Livak or DNAzol methods |
P. vivax, P. falciparum, P. ovale, P. malariae
| 294 | 0.2 ng–0.2 pg | |
P. vivax, P. falciparum, P. ovale, P. malariae
| 205 | 2 ng–4 pg | ||
18s-rRNA Taqman assay [4] |
P. falciparum, P. ovale, P. malariae, P. vivax
| 47 | 0.2 pg | |
P. falciparum
| 60* | 2 pg | ||
Cytochrome B single PCR [22] | IsoQuick nucleic acid extraction kit |
P. vivax, P. falciparum
| 96 | 0.2 pg |
DHFR-TS nested [30] | Chelex |
P. falciparum
| >294 | 4–40 pg |
Fast COX-I single PCR [this manuscript] | CTAB |
P. vivax, P. falciparum, P. ovale s.l., P. knowlesi, P. ovale wallikeri
| 62 | 0.043 pg |
PCR technique | Required PCR reagents | Estimated cost of the PCR kit | µL of polymerase used per reaction | Cost of PCR diagnosis per sample | Cost of 2122 reactions for Plasmodium detection |
---|---|---|---|---|---|
18s-rRNA nested PCR | Taq polymerase kit (Invitrogen) | ~210 USD | 0.1 µL for nest-1 and 0.1 µL nest-2 | ~0.42 USD | ~892 USD |
Conventional COX-I single PCR | Taq polymerase kit (Invitrogen) | ~210 USD | 0.2 µL in a single reaction | ~0.42 USD | ~892 USD |
Fast COX-I single PCR | Blood Phusion polymerase kit (Thermo) | ~418 USD | 0.125 µL in a single reaction | ~0.26 USD | ~552 USD |