A novel nested polymerase chain reaction assay targeting Plasmodium mitochondrial DNA in field-collected Anopheles mosquitoes.
Med Vet Entomol
; 32(3): 372-377, 2018 09.
Article
en En
| MEDLINE
| ID: mdl-29344968
ABSTRACT
Sensitive techniques for the detection of Plasmodium (Aconoidasida Plasmodiidae) sporozoites in field-collected malaria vectors are essential for the correct assessment of risk for malaria transmission. A real-time polymerase chain reaction (RT-PCR) protocol targeting Plasmodium mtDNA proved to be much more sensitive in detecting sporozoites in mosquitoes than the widely used enzyme-linked immunosorbent assay targeting Plasmodium circumsporozoite protein (CSP-ELISA). However, because of the relatively high costs associated with equipment and reagents, RT-PCRs are mostly used to assess the outcomes of experimental infections in the frame of research experiments, rather than in routine monitoring of mosquito infection in the field. The present authors developed a novel mtDNA-based nested PCR protocol, modified from a loop-mediated isothermal amplification (LAMP) assay for Plasmodium recognition in human blood samples, and compared its performance with that of routinely used CSP-ELISAs in field-collected Anopheles coluzzii (Diptera Culicidae) samples. The nested PCR showed 1.4-fold higher sensitivity than the CSP-ELISA. However, nested PCR results obtained in two laboratories and in different replicates within the same laboratory were not 100% consistent, probably because the copy number of amplifiable Plasmodium mtDNA was close in some specimens to the threshold of nested PCR sensitivity. This implies that Plasmodium-positive specimens should be confirmed by a second nested PCR to avoid false positives. Overall, the results emphasize the need to use molecular approaches to obtain accurate estimates of the actual level of Plasmodium circulation within malaria vector populations.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Plasmodium
/
Ensayo de Inmunoadsorción Enzimática
/
ADN Protozoario
/
Reacción en Cadena en Tiempo Real de la Polimerasa
/
Anopheles
Límite:
Animals
Idioma:
En
Revista:
Med Vet Entomol
Asunto de la revista:
BIOLOGIA
/
MEDICINA VETERINARIA
Año:
2018
Tipo del documento:
Article
País de afiliación:
Italia