Nucleic Acid Extraction Techniques - PMC

Since thermostable Taq DNA polymerase was discovered in , nucleic acid amplification techniques have made great strides and contributed greatly to progress in the life sciences. These techniques were introduced into the clinical laboratory and have produced great changes in diagnostic tools and tests. In particular, there have been many innovative molecular testing developments in the field of diagnostic microbiology.

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Introduction

Since thermostable Taq DNA polymerase was discovered in , nucleic acid amplification techniques have made great strides and contributed greatly to progress in the life sciences. These techniques were introduced into the clinical laboratory and have produced great changes in diagnostic tools and tests. In particular, there have been many innovative molecular testing developments in the field of diagnostic microbiology.

Culture methods of bacterial identification are labor-intensive and time-consuming. However, they are simple, cheap, and remain the gold standard. It also is possible to perform antimicrobial susceptibility testing on cultured isolates, so conventional culture methods with biochemical phenotyping are still the most common procedures performed in clinical microbiology laboratories [1]. To further assist in microbial identification, nucleic acid amplification has been introduced in the clinical microbiology laboratory. Such testing was initially done for viruses, allowing detection of small amounts of viral nucleic acid quickly. Similar tests also have been applied to bacteria, especially those that require cell cultures, are difficult to grow on routine culture media, or are slow growing such as Chlamydia, Neisseria gonorrhoeae, and Mycobacterium. In addition, there are ongoing attempts to apply these new techniques for routine clinical microbiology testing, including the diagnosis of sepsis [1].

The development of nucleic acid amplification has proceeded at an unprecedented pace and achieved higher sensitivity and specificity [2]. However, in order to obtain satisfying results with this new technique, the testing must go through several important steps. Preanalytical testing variables comprise sample collection and preparation, specimen transport and storage, stability of the nucleic acid in the samples, and nucleic acid extraction [3, 4].

Nucleic acid extraction is the first step of any amplification experiment no matter what kind of amplification is used to detect a specific pathogen [1, 5]. It is a crucial preanalytic step in the development and performance of any successful molecular diagnostic method and ensures a reliable result [3, 4]. We must pay attention to the technical progress of the nucleic acid extraction as well as to the method for amplification and detection of nucleic acids in order to obtain satisfactory results. Nucleic acid extraction consists of three major processes: isolation, purification, and concentration. Commercial extraction kits are commonly used in the clinical microbiology laboratory [2]. These kits provide the essential requirements for nucleic acid extraction. These essential requirements have been well described by Boom et al. [6, 7] as follows: Extraction should be simple, rapid, and should show high sensitivity and specificity. It is preferred that there be no requirements for specialized equipment or special knowledge and skills. The final nucleic acid should be pure and easy to modify for various amplification techniques. The reagents and their product should be harmless, and the process of preparation should resist contamination with other specimens. If the final volume of eluate is small, detection limits are maximized. When we deal with clinical specimens, we also should consider the elimination of potential inhibitors of the DNA polymerase and the removal of pathogenicity from hazardous pathogens as well as good target recovery and establishment of the integrity of nucleic acid targets [2]. Ideally, the final target is pure nucleic acid without amplification inhibitors or contaminants such as protein, carbohydrate, and other nucleic acids [8].

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There are a few points to be specially considered when we consider the use of nucleic acid extraction in the field of clinical microbiology. Usually, we extract DNA or RNA, and some times, we may extract both, depending on the circumstances. The targets for nucleic acid extraction are diverse. They can be the cultured bacterial isolates themselves. Alternatively, we can use culture media, including blood culture bottles or various clinical specimens such as sputum, stool, urine, tissue, or cerebrospinal fluid [1]. In terms of nucleic acid extraction, they may target the same nucleic acid, but they have different implications for the extraction procedure itself. Nucleic acid extraction from cultured bacteria is relatively simple because they are pure colonies and they contain large numbers of organisms. However, we need to recognize that gram-positive bacteria have thick walls, such that nucleic acid extraction is more difficult than it is from gram-negative bacteria, which have thinner walls [5]. For clinical specimens, the details of the method depend on the characteristics of each specimen. It is important to remember that the subject is nucleic acid, not of humans, but of bacteria, virus, or fungus. If we extract from clinical specimens containing human cells, we cannot help mixing human DNA and sometimes, recovery of microbial DNA can be diminished by the presence of human DNA.

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