Specimen Processing

Extraction of nucleic acids from biological materials is one of the most important steps performed in the molecular virology laboratory. High quality nucleic acids are required for most applications, but they are especially important when nucleic acid amplification and sequencing are contemplated. The purposes of specimen processing steps are (1) to make the nucleic acids available for amplification, hybridization, or detection; (2) to concentrate the nucleic acids; and (3) to remove any inhibitory substances that might be present in the specimen.

Molecular diagnostic methods can be inhibited by (1) chelating divalent cations such as Mg2+, (2) degradation of the target and/or primer nucleic acids, or (3) inactivation of the enzymes used in these procedures. Several reports have demonstrated that heme (Mercier et al., 1990; Ruano et al., 1992), heparin (Beutler et al., 1990; Holodniy et al., 1991), phenol (Katcher and Schwartz, 1994), polyamines (Ahokas and Erkkila, 1993), plant polysaccharides (Demeke and Adams, 1992), and urine (Kahn et al., 1991) can inhibit polymerase chain reactions (PCR). Unfortunately, little is known about the substances that inhibit other enzymes used in molecular diagnostic procedures. Specimen processing procedures must therefore remove these substances without degrading the nucleic acid target or adding any other inhibitory substances.

The most conventional methods for extracting nucleic acids from clinical specimens involve proteinase K digestion(s) followed by multiple phenol and chloroform:isoamyl alcohol (24:1) extractions. The resulting nucleic acids are precipitated in the presence of salts and cold ethanol. The DNA pellet is washed with cold 70% ethanol to remove any contaminants, dried, and dissolved in a suitable buffer system for the ensuing procedures. Although these procedures have proven useful for extracting genomic DNA from tissues, they are often too lengthy and laborious for routine use in a molecular virology laboratory that only tests spinal fluids. In addition, multiple chloroform extraction of specimens containing low copy numbers of viral DNAs can produce false negative reactions because of sample loss. The use of copious amounts of phenol in the laboratory is often undesirable because of the caustic and poisonous nature of these chemicals. The nonorganic salting-out procedures of Miller and Polesky (1988) and Buffone and Darlington (1985) provide alternatives to organic chemical extractions. In general, extraction procedures must be tailored to the individual specimen type and to the suspect agent. Viruses present in high concentrations in highly inhibitory substances (e.g., rotavirus in fecal specimens) can be extracted extensively before performing nucleic acid testing. Viruses that are present in low copy numbers [e.g., herpes simplex virus (HSV) in vitreous or spinal fluids] should be handled as little as possible to prevent nucleic acid loss.

Specimen storage time and temperatures can have a significant impact on nucleic acid recovery and the efficiency of subsequent diagnostic procedures. Lysis of red blood cells can influence PCR reactions by inhibition of Taq DNA polymerase by heme. Heme can also bind to and damage DNA at the elevated temperatures used in PCR reactions (Winberg, 1991). In addition, lysis of granulocytes releases proteases and nucleases that degrade viral particles and nucleic acids. Cuypers et al. (1992) reported a 1000- to 10,000fold reduction in hepatitis C virus (HCV) RNA concentrations when whole blood and serum were stored at room temperature. However, degradation was even faster when whole blood was stored at 4°C, presumably because of increased granulocyte lysis at 4°C relative to room temperature. The recommended method for storing specimens for HCV testing is allowing the blood to clot, removing the serum, and storing the serum at 4°C or -20°C.

The best DNA yields and diagnostic results are achieved when the nucleic acids are extracted from fresh specimens. Once the nucleic acids are purified and precipitated in ethanol, they are stable at -20°C for years. If shipping whole blood is absolutely necessary [e.g., for human immunodeficiency virus (HIV) testing], specimens should be sent on wet ice and the nucleic acids should be extracted as soon as the samples are received in the reference laboratory (Cushwa and Medrano, 1993). Minimizing exposure of blood samples to temperatures >23°C is also important because of decreased DNA yields from specimens stored at these temperatures (Cushwa and Medrano, 1993).

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