Month: April 2021

Molecular diagnostic testing techniques using nucleic acids were pioneered through polymerase chain reaction (PCR), which is the predominant technique due to its familiarity, sensitivity, and robustness.

What does polymerase chain reaction mean, and why is everyone talking about it?

Polymerase chain reaction is a technique used to “amplify” or make many copies of small segments of DNA, allowing scientists to make extensive genetic research and clinicians to detect a variety of diseases. It is extremely useful in diagnosis of genetic disorders, DNA fingerprinting and detection of viruses or bacteria. Yet, the increasing use of molecular diagnostic techniques has underlined simplicity and speed as key factors for adoption, especially in small-scale diagnostics settings.

For field applications and point-of-care, isothermal amplification techniques could successfully fulfill these criteria. In isothermal amplification methods, there is no need for expensive thermal cycling equipment. In fact, isothermal amplification methods require only a single temperature. Therefore, amplification reactions could be accomplished in only 5 minutes. 

Applications of isothermal amplification techniques

Loop-mediated isothermal amplification (LAMP) is the most widespread isothermal method. LAMP has been applied to diagnostic detection of pathogens in food, animal, plant, and clinical samples. For example, in 2011, Zhang and colleagues used LAMP to detect Salmonella spp. with high sensitivity in vegetables and similarly. In the following year, Wang and colleagues analyzed Shiga-toxin producing E. coli (STEC) using LAMP assays in ground beef, spinach, beef trim, and lettuce.

Principle of loop-mediated isothermal amplification (LAMP)

Loop-mediated isothermal amplification, which is based on auto cycling strand displacement, consists of two main steps:

1. In the first step, four primers are specifically designed to form a dumbbell‐shaped structure.
2. In the second step, this dumbbell‐shaped structure is utilized as a template for further amplification cycles which proceed with self‐primed DNA synthesis with the two inner primers.

The two inner primers for LAMP are designed in a way that a looped structure should be formed every time a single‐stranded DNA is produced. These two inner primers consist of two sets of sequences that can bind both sense and antisense strands, with a TTTT spacer between the sets of sequence.

The two outer primers help in initiating strand displacement DNA synthesis to make the loop structure at one end. As a way to enhance the reaction speed, two optional primers, LF (loop forward) and LB (loop backward), could also be added to the amplification reaction.

Advantages of LAMP

Loop-mediated isothermal amplification has exceptionally high specificity because a set of four primers with six binding sites must hybridize correctly to their target sequence before biosynthesis of DNA. Its low-cost, simplicity, flexibility, high selectivity and sensitivity makes LAMP a popular platform for molecular diagnostics. 

In 2015, Zhang and colleagues used LAMP successfully for the rapid diagnosis of pathogenic bacteria in clinical sputum specimens of acute exacerbation of chronic obstructive pulmonary disease, indicating the high sensitivity of LAMP compared to that of sputum culturing methods.

Furthermore, LAMP is simple and easy to perform, requiring only a DNA polymerase, four primers and a heat block or regular laboratory water bath for reaction. Recently, because of the coronavirus outbreak, LAMP, combined with reverse transcription is currently used as one of the leading methods in the amplification of RNA sequences, performing with high efficiency.

Sources:

Tanner, Nathan A., and Thomas C. Evans Jr. “Loop‐mediated isothermal amplification for detection of nucleic acids.” Current protocols in molecular biology 105.1 (2014): 15-14.

Notomi, Tsugunori, et al. “Loop-mediated isothermal amplification of DNA.” Nucleic acids research 28.12 (2000): e63-e63.

Dolka, Beata, Agata Anna Cisek, and Piotr Szeleszczuk. “The application of the loop-mediated isothermal amplification (LAMP) method for diagnosing Enterococcus hirae-associated endocarditis outbreaks in chickens.” BMC microbiology 19.1 (2019): 1-13.

Zhang, Guodong, Eric W. Brown, and Narjol González-Escalona. “Comparison of real-time polymerase chain reaction, reverse transcriptase real-time polymerase chain reaction, loop-mediated isothermal amplification, and the FDA conventional microbiological method for the detection of Salmonella spp. in produce.” Applied and environmental microbiology 77.18 (2011): 6495-6501.

Wang, Fei, et al. “Rapid and specific detection of Escherichia coli serogroups O26, O45, O103, O111, O121, O145, and O157 in ground beef, beef trim, and produce by loop-mediated isothermal amplification.” Applied and environmental microbiology 78.8 (2012): 2727-2736

Zhang, Wei, et al. “Application of loop-mediated isothermal amplification (LAMP) assay for the rapid diagnosis of pathogenic bacteria in clinical sputum specimens of acute exacerbation of COPD (AECOPD).” International journal of clinical and experimental medicine 8.5 (2015): 7881.

The extraction of nucleic acids is the first step in numerous research and clinical applications.¹ High-throughput multiplex systems have been introduced for genotyping, gene expression, and epigenetic profiling that require the extraction of numerous nucleic acid samples as starting material. The results obtained using these approaches have broad implications both for basic research and for the diagnosis and follow up of clinical cases.

Manual procedures for nucleic acid extraction are well established but are time and effort consuming.² Thus, there is an urgent need to develop optimized methods for nucleic acid extraction from clinical and research samples, that require less effort and time of the laboratory personnel. Automated extraction platforms have undergone rapid development and represent an alternative to manual extraction methods.^3,4,5 However, their optimization also faces certain challenges that still need to be addressed.

Quality standards for automated extraction platforms

Throughout the process of their development, automated extraction platforms are rigorously tested for the linearity and reliability of extraction.^3,4,5 Moreover, their extraction yield is quantified and compared with the yield of manual extraction procedures. Further, the purity of the extracted nucleic acids, which is necessary to ensure their proper performance in downstream applications, is tested.²

Advancements in the development of automated extraction platforms

The establishment of automated nucleic acid extraction platforms, liquid handling systems, and microfluidic chips has advanced nucleic acid extraction both for research and clinical applications. 

Automated nucleic acid extraction platforms

Several automated nucleic acid extraction platforms based mainly on solid-phase or magnetic beads methods have been developed.^1,3,4,5 Major advantages of the introduced in the practice automated extraction platforms include the speed of extraction and the opportunity to process large batches of samples simultaneously. However, potential tradeoffs may be possible issues with nucleic acid purity and the time needed to set up the extraction system and samples.⁶

Automated liquid handling systems

Liquid handling is a challenging aspect in the process of nucleic acid extraction.² Therefore, automated liquid handling systems have also been developed and routinely implemented in research and clinical laboratories. These liquid handling robots dispense precise volumes of reagents or samples and enable the simultaneous handling of multiple samples and their rapid processing. Barcode readers are an important part of such systems since they ensure the traceability of the samples. Overall, automated liquid handling systems facilitate the automation of different laboratory procedures, including nucleic acid extraction.¹

Microfluidic chips

Nucleic acid extraction generally requires specialized equipment, dedicated facilities, and trained personnel, which may not be readily available at certain locations. However, the speed with which nucleic acid extraction and downstream applications can be performed may be very important, particularly for the analysis of clinical samples. The development of point-of-care platforms may resolve this problem by performing nucleic acid extraction and downstream applications without the need for specialized facilities and at the location where the clinical decisions are made. As a step in this direction, microfluidic chips that are based on solid phase extraction and contain all necessary reagents on inner chambers have been introduced.¹ However, they still do not fulfill the requirements for a point-of-care extraction method particularly because the currently existing procedures for sample preparation are not suited to the point-of-care environment.¹

Trends in the development of automated extraction platforms

Automated platforms for the extraction of nucleic acids have found broad applications in research and clinical laboratories. Future directions in their development include the miniaturization of the devices and the simplification of their handling. Moreover, the development of extraction systems that can be used as point-of-care devices is of particular interest due to their potential important clinical implications, especially in resource-limited settings.

Sources:

1. Ali N, Rampazzo RCP, Costa ADT, Krieger MA. Current nucleic acid extraction methods and their implications to point-of-care diagnostics. Biomed Res Int. 2017;2017:9306564.
2. Tan SC, Yiap BC. DNA, RNA, and protein extraction: the past and the present. J Biomed Biotechnol. 2009;2009:574398. doi: 10.1155/2009/574398. Erratum in: J Biomed Biotechnol. 2013;2013:628968.
3. Verheyen J, Kaiser R, Bozic M, Timmen-Wego M, Maier BK, Kessler HH. Extraction of viral nucleic acids: comparison of five automated nucleic acid extraction platforms. J Clin Virol. 2012;54(3):255-259.
4. Perandin F, Pollara PC, Gargiulo F, Bonfanti C, Manca N. Performance evaluation of the automated NucliSens easyMAG nucleic acid extraction platform in comparison with QIAamp Mini kit from clinical specimens. Diagn Microbiol Infect Dis. 2009;64(2):158-165.
5. Lewandowski K, Bell A, Miles R, Carne S, Wooldridge D, Manso C, Hennessy N, Bailey D, Pullan ST, Gharbia S, Vipond R. The effect of nucleic acid extraction platforms and sample storage on the integrity of viral RNA for use in whole genome sequencing. J Mol Diagn. 2017;19(2):303-312.
6. Thatcher SA. DNA/RNA preparation for molecular detection. Clin Chem. 2015;61(1):89-99.