Application of Nucleic Acid Extraction in Clinical Specimens
What is the Nucleic acid extraction from clinical specimens?
Nucleic acid extraction from clinical specimens is quite different from that from cultured isolates of bacteria or fungi. This extraction step can influence the subsequent performance of the diagnostic tests; the efficiency of nucleic acid extraction is related directly to the sensitivity of the final test results. Each clinical specimen has diverse characteristics. Blood and stool are composed of many substances, and among these, heme and bile act as inhibitors of amplification and should be removed. We can find the comparison results for nucleic acid extraction; however, this cannot ensure that we can adapt this result to different specimens and pathogens. In previous reports, herpes simplex virus DNA was isolated relatively easily from genital swabs, but obtaining bacterial DNA from stool samples was more complex.
To overcome these limitations, the extraction method must be evaluated before routine testing of specific pathogens from specific specimens. For detection of clinically important viruses, extraction efficiency was evaluated in various specimens, including serum, urine, and cerebrospinal fluid, and good performance was confirmed. However, we should not extrapolate these specific results to all types of virus and specimens.
Cellular Component
Tissue is an important clinical specimen for diagnosing localized cytomegalovirus infections in a transplanted organ as biopsy is a common method used to evaluate potential CMV infection. However, tissue specimens have problems because the large amount of human tissue contains cellular DNA, proteins, and other materials. So, a more complex step to extract the nucleic acid of the microbial pathogen is needed. Most commercial kits for tissue specimens extract human nucleic acid also. In recent years, we have been able to extract the viral nucleic acid from clinical specimens having cellular components, and there have been a few trials of these kits to detect various clinically important viruses.
Serum, Plasma, and Whole Blood
Clinicians place great emphasis on the detection of bacteria and fungi in blood. Therefore, nucleic acid extraction from blood has become very important. Many researchers have found that there are numerous PCR inhibitors in blood culture bottles such as sodium polyanetholesulfonate (SPS) and hemins. Millar and collaborators compared several commercial and in-house extraction methods used to detect bacteria and fungi in BacT/Alert blood culture bottles . To reduce the detection time, the serum, plasma, or whole blood is used as a main specimen for detection of bacteria and fungi. Serum or plasma is more efficient and convenient than whole blood because whole blood includes many PCR inhibitors. Most commercial kits showed a high recovery rate of pathogen DNA, but only those methods that used heat lysis with an alkali wash could remove PCR inhibitors. Detection of brucellosis was highly sensitive even though Brucellae are facultative intracellular pathogens. Similarly, kits containing proteinase K showed better yield of Brucella in serum specimens. However, to enhance the sensitivity of PCR amplification, whole blood is considered as a final target because it contains more pathogens than serum or plasma. Although the nucleic acid kits were not developed to extract microbial DNA from whole blood, all commercial kits are able to do so. In recent years, we have been able to use several automated systems to extract bacterial or fungal DNA from whole blood although they are expensive. They are suitable for high-throughput detection.
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