The use of molecular methods does not depend on the isolation or growth of the pathogen or the detection of an immune response against the agent. And yes, the genetic information contained in the samples. For this reason, molecular diagnosis is making great progress in the investigation of infectious, genetic and oncological diseases.
What allowed this growth was, mainly, the expansion of knowledge about the genomes of viruses, bacteria and fungi combined with advances in bioinformatics, which allowed analysis of nucleic acid sequences to find the best regions for diagnosis.
Among the techniques described in the literature, the most prominent is the polymerase chain reaction ( polymerase chain reaction , PCR) . This technique is used for the selective amplification of a specific region of a target DNA molecule, in which only a single DNA molecule can serve as a template for amplification to produce thousands of copies.
Although the sensitivity of PCR is the differential of the technique, some factors can compromise the result. Contamination is a frequent concern. The DNA molecules previously amplified in other reactions (amplicons) are the ones that pose the greatest contamination threats. For this reason, it is essential to adopt practices that ensure its efficiency and reliability of results, especially when used for diagnostic purposes. The quality and quantity of the extracted nucleic acids are greatly affected by the collection of the sample, its handling and transport and the choice of the extraction method. Thus, it becomes relevant to understand the possible causes of error, in the pre-analytical phase, which are more frequent in molecular diagnosis.
Blood samples and bone marrow aspirate
Studies indicate that heparin and heme are potent inhibitors of the polymerase chain reaction (PCR), thus the recommended anticoagulants for these samples are ethylene diaminetetraacetic acids (EDTA) and citrate dextrose (ACD).
Whole blood is stable at room temperature for 24 hours for DNA analysis and up to eight days when cooled (2-8 ° C). For cellular RNA analysis, blood must be collected with a stabilizing additive. Namely, collection and storage of whole blood without stabilizer is not recommended for analysis of genetic transcription, due to the induction and degradation of RNA that occurs ex vivo .
Bone marrow must be collected using a syringe with EDTA, and the team responsible for processing must be notified as soon as the sample arrives at the laboratory. For DNA extraction, bone marrow aspirate can be stored temporarily for up to 72 hours at 2-8 ° C before processing. However, if it is necessary to store for longer than that, the erythrocytes must be removed and the sample must be frozen at -20 ° C (valid for several months).
Bone marrow sample collection © 2019 Merck Sharp & Dohme Corp., subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA
- Caution:erythrocyte removal can release heme and inhibit the PCR reaction
CEREBROSPINAL FLUID (CSF) SAMPLE
CSF samples must be transported at 2-8 ° C for DNA analysis. If they cannot be processed immediately, they can be frozen (at -20 ° C or below) to search for DNA viruses (HSV, CMV and VZV).
Circulation of LCR – OpenStax [CC BY 4.0 (https://creativecommons.org/licenses/by/4.0)]
For RNA analysis (including RNA viruses, such as enteroviruses), the sample must be placed in a bath with crushed ice and the RNA extracted within four hours of collection. If this is not possible, remove possible contamination with erythrocytes and freeze the sample, transporting it with dry ice.
Image: Minutes of Health Sciences, São Paulo, Vol.4, N ° .3, p. 1-24, JUL-SET 2016
Sputum for DNA analysis should be collected in a sterile flask and transported to the laboratory at room temperature, if it takes up to 30 minutes; otherwise, it must be refrigerated. Sputum DNA can be stable for up to one year when frozen at -70 ° C. Some DNA extraction protocols use sputum concentration to increase the sensitivity of screening for infectious agents, and the manufacturer’s recommendations should be followed in these cases. .
T1 Lysis Buffer
The extraction of DNA and / or RNA is the first step in the execution of different procedures in Molecular Biology. Thus, the choice of the best products for this stage is fundamental for the final quality of the diagnosis.
Lysis Buffer T1 is Kasvi’s novelty in the reagent line. It is the ideal tool for a simple, fast and efficient manual extraction and purification of different types of samples. In addition to providing up to 13 additional extraction protocols.