INSTITUTO DE INVESTIGACIONES EN MICROBIOLOGIA Y PARASITOLOGIA MEDICA
Unidad Ejecutora - UE
An improved DNA isolation technique for PCR detection of Strongyloides stercoralis in stool simples.
REPETTO S; ; C. D. ALBA SOTO; ; S. I. CAZORLA; ; M. L. TAYELDIN;; S. CUELLO;; M.B. LASALA;; V.S. TEKIEL; S.M. GONZÁLEZ CAPPA.
ELSEVIER SCIENCE BV
Lugar: Amsterdam; Año: 2013 vol. 126 p. 110 - 110
Strongyloides stercoralis is a nematode that causes severe infections in immunocompromised patients.The low parasitic burden of chronically infected patients makes diagnosis difficult to achieve by conventionalmethods. Here, an in-house (IH) method for the isolation of parasite DNA from stools and aPCR assay for the molecular diagnosis of S. stercoralis were optimized. DNA yield and purity improvedwith the IH method which included a step of incubation of stool samples with a glycineSDS buffer andmechanical disruption prior to DNA extraction. For the PCR assay, the addition of bovine serum albuminwas required to neutralize inhibitors present in stool. The analytical sensitivity of the PCR using DNA astemplate, isolated with the IH method, was superior to the commercial one. This study demonstrates thata combined method that adds the step of glycineSDS buffer incubation plus mechanical disruption priorto DNA isolation with the commercial kit increased PCR sensitivity to levels of the IH method. Finally, ourassay was tested on 17 clinical samples. With the IH method for DNA isolation, a S. stercoralis specific bandwas detected by PCR in the first stool sample in all patients (17/17), while with the commercial kit, our S.stercoralis-specific band was only observed in 7 samples. The superior efficiency of the IH and combinedmethods over the commercial kit was demonstrated when applied to clinical samples with low parasiticburden. These results show that the DNA extraction procedure is a key to increase sensitivity of the S.stercoralis PCR assay in stool sa