The amplification conditions were modified and amplification time substantially reduced from six to two hours.
The new cycling parameters were: step 1, 95°C for 15 min; step 2, denaturation at 95°C for 10 sec; step 3, annealing at 65°C for 10 sec; step 4, extension at 72°C for 30 sec; steps 2-4 repeated 45 times; and step 5, 72°C for 10 min.
Nested PCR is a commonly used technique in diagnosis of malaria owing to its high sensitivity and specificity.
However, it is time-consuming, open to considerable risk of contamination and has low cost-efficiency.
All three assays detected as low as 0.05 p/μl, though not consistently.Both sensitivity and specificity of the novel mitochondrial PCR was 100% and proved non-inferior to that of the reference nested PCR. dating dk login Brønderslev Sequencing of genus-specific mitochondrial PCR products could be used for species determination.].Using amplification targets presented in multiple copies, such as r RNA 18S, or mitochondrial targets with an even higher copy number, might increase sensitivity.genus-specific single-round amplification PCR programmes, based on previously published primers targeting 18S and mitochondrial genome, were compared with a widely used nested 18S PCR.
The mitochondrial PCR detected one additional sample, also positive by microscopy, and was the only method with 100% sensitivity (28/28).The sensitivity and specificity of the mitochondrial PCR were statistically non-inferior to that of the reference nested PCR.Analyses of dilution series from reference material were performed, as well as retrospective analyses of 135 blood samples, evaluated by routine microscopy, from 132 fever patients with potential imported malaria.Sequencing of the 220 bp mitochondrial PCR products was performed.Microscopy missed two infections detected by all PCR assays.
Sequencing of the genus-specific mitochondrial PCR products revealed different single nucleotide polymorphisms which allowed species identification of the 28 sequences with following distribution; 20 In this study, design of PCR programmes with suitable parameters and optimization resulted in simpler and faster single-round amplification assays.
The amplifications were performed by using Gene Amp PCR System 9700 (Applied Biosystems, Carlsbad, CA, USA), and the PCR products were analysed by electrophoresis using 2% Sea Kem® agarose gel (Lonza, Rockland, ME, USA) with 1X Gel Red™ (Biotium, Hayward, CA, USA).
Concentrations of primers and additional Mg Cl] to a single-round amplification assay with cycle parameters as follows: step 1, 95°C for 15 min; step 2, denaturation at 95°C for 10 sec; step 3, annealing at 63°C for 10 sec; step 4, extension at 72°C for 75 sec; steps 2-4 repeated 50 times; and step 5, 72°C for 10 min.].
In comparison, a LAMP assay employing primers targeting 18S had a sensitivity limited to approximately 100 copies of the gene for The patient material used in this study had been collected between 20 at Haukeland University Hospital, Bergen, Norway.
It included 135 whole blood samples from a cohort of 132 fever patients with potential imported primary or recurrent malaria.