Improved DNA extraction technique from clot for the diagnosis of Chagas disease
| Autor: | Alejandra Pando, Jorge Flores, Rony Colanzi, Manuela Verastegui, Yomara K. Romero, Bolivia, Freddy Tinajeros, Josephine Henderson-Frost, Ricardo Bozo, Robert H. Gilman, Caryn Bern, Richard Lerner, Holger Mayta |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Chagas disease protozoal DNA parasitology Physiology RC955-962 serology Artificial Gene Amplification and Extension Buffy coat Pathology and Laboratory Medicine Polymerase Chain Reaction law.invention Serology 0302 clinical medicine law Arctic medicine. Tropical medicine Medicine and Health Sciences Medicine genetics DNA extraction Polymerase chain reaction Whole blood Protozoans Eukaryota Body Fluids 3. Good health female Blood Infectious Diseases Real-time polymerase chain reaction Molecular Diagnostic Techniques diagnostic test real time polymerase chain reaction blood clot blood sampling Anatomy Public aspects of medicine RA1-1270 purl.org/pe-repo/ocde/ford#3.03.06 [https] Research Article Neglected Tropical Diseases Trypanosoma Trypanosoma cruzi 030231 tropical medicine satellite DNA guanidine Real-Time Polymerase Chain Reaction Sensitivity and Specificity Article 03 medical and health sciences Extraction techniques blood Diagnostic Medicine molecular diagnosis Parasitic Diseases Humans controlled study Chagas Disease Serologic Tests human procedures Molecular Biology Techniques Molecular Biology hemagglutination test kit Protozoan Infections isolation and purification Diagnostic Tests Routine business.industry Organisms Public Health Environmental and Occupational Health Biology and Life Sciences DNA Protozoan Tropical Diseases medicine.disease major clinical study Molecular biology Parasitic Protozoans enzyme linked immunosorbent assay Research and analysis methods 030104 developmental biology hemagglutination test business blood clot lysis Blood sampling |
| Zdroj: | PLoS Neglected Tropical Diseases, Vol 13, Iss 1, p e0007024 (2019) PLoS Neglected Tropical Diseases |
| ISSN: | 1935-2735 1935-2727 |
| Popis: | Background The detection of Trypanosoma cruzi genetic material in clinical samples is considered an important diagnostic tool for Chagas disease. We have previously demonstrated that PCR using clot samples yields greater sensitivity than either buffy coat or whole blood samples. However, phenol-chloroform DNA extraction from clot samples is difficult and toxic. The objective of the present study was to improve and develop a more sensitive method to recover parasite DNA from clot samples for the diagnosis of Chagas disease. Methodology/Principal findings A total of 265 match pair samples of whole blood–guanidine (GEB) and clot samples were analyzed; 150 were from Chagas seropositive subjects. DNA was extracted from both whole blood-guanidine samples, using a previously standardized methodology, and from clot samples, using a newly developed methodology based on a combination of the FastPrep technique and the standard method for GEB extraction. A qPCR targeting the nuclear satellite sequences was used to compare the sample source and the extraction method. Of the 150 samples from Chagas positive individuals by serology, 47 samples tested positive by qPCR with DNA extracted by both GEB and clot, but an additional 13 samples tested positive only in DNA extracted from clot. No serology-negative samples resulted positive when tested by qPCR. Conclusions The new methodology for DNA extraction from clot samples improves the molecular diagnosis of Chagas disease. Author summary Detection of nucleic acid has become an important tool for the diagnosis of Chagas disease. Whole blood samples are usually the source of DNA and qPCR the preferred technique to demonstrate the presence of T. cruzi DNA. Although DNA extracted from clot samples has shown higher sensitivity than from whole blood, DNA extraction is performed using phenol-chloroform, which has biohazard issues. We theorize that a clot traps parasites, making it a better source of DNA for Chagas diagnosis using PCR. The present study describes a new DNA extraction methodology from clot samples which avoids the use of phenol-chloroform. The new methodology was compared to the internationally standardized diagnostic method, which is based on extraction of DNA from whole blood preserved with guanidine EDTA and a commercial kit. |
| Databáze: | OpenAIRE |
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