STANDARDIZATION OF THE PCR TECHNIQUE FOR THE DETECTION OF THE ITS-1 SEQUENCE OF NECATOR AMERICANUS (STILES, 1902) AND CLONING OF THE PRODUCT FOR USE AS A CONTROL

Abstract

Hookworm is generally caused by Necator americanus (Stiles, 1902), causing digestive symptoms and anemia. The diagnosis by coprology can have low sensitivity, especially in light parasite loads. The Polymerase Chain Reaction (PCR) is a sensitive and specific technique, which must be adapted to laboratory conditions and positive controls are necessary. The objective of this work was the standardization of the PCR technique for the amplification of the ITS-1 sequence of N. americanus in stool samples and its cloning for its use as a control. Three DNA extraction protocols were standardized (Phenol/chloroform, Saline Precipitation, and Chelex® 100 Resin). Optimal reagent concentrations (MgCl , BSA, dNTP, primers, and Taq polymerase) were determined, as well as the hybridization 2 temperature and number of cycles. The analytical sensitivity and specificity of the technique was determined. For cloning, the ITS-1 sequence amplified by PCR was purified and ligated with the vector pGEM-T-Easy. Competent E. coli XL1Blue MRF` cells were transformed with the ligation mixture (pGEM-T-easy-Na-ITS1), recombinant colonies were identified and plasmid DNA was extracted from them. The best DNA extraction protocol was phenol / chloroform, the optimal conditions for PCR were; 1.5 mM MgCl , 0.5 mg/mL BSA, 100 μM dNTP, 0.6 μM primers, and 1 U Taq polymerase, 56 °C 2 hybridization temperature and 40 cycles. The optimal amounts of reagents were less than the amounts used by other authors, allowing saving of reagents. 100% specificity and an analytical sensitivity of 10 pg of DNA (extracted from stool samples), and 10 ag of the plasmid (with the cloned sequence) were obtained, which worked very well as positive control.