[21] Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction
Publisher Summary
This chapter discusses the specific synthesis of deoxyribonucleic acid (DNA) in vitro through the medium of a polymerase-catalyzed chain reaction. A method whereby a nucleic acid sequence can be exponentially amplified in vitro is described in the chapter. The same method can be used to alter the amplified sequence or to append new sequence information to it. It is necessary that the ends of the sequence be known in sufficient detail that oligonucleotides can be synthesized, which will hybridize to them and that a small amount of the sequence be available to initiate the reaction. The oligonucleotides are complementary to different strands of the desired sequence and at relative positions along the sequence such that the DNA polymerase extension product of the one, when denatured, can serve as a template for the other and vice versa. Oligonucleotides were synthesized using an automated DNA synthesis machine (Biosearch, Inc., San Rafael, California) using phosphoramidite chemistry. “Mispriming"” can be usefully employed to make intentional in vitro mutations or to add sequence information to one or both ends of a given sequence. The chapter explores the possibility of utilizing a heat-stable DNA polymerase so as to avoid the need for addition of new enzyme after each cycle of thermal denaturation
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Rapid and sensitive detection of nucleic acids using an RAA-CRISPR/Cas12b one-pot detection assay (Rcod)
2024, TalantaRapid, sensitive and specific methods are crucial for nucleic acid detection. CRISPR/Cas12b has recently been widely used in nucleic acid detection. However, due to its thermophagic property, DNA isothermal recombinase-aided amplification (RAA) and subsequent CRISPR/Cas12b detection require two separate reactions, which is cumbersome and inconvenient and may cause aerosol pollution. In this study, we propose an RAA-CRISPR/Cas12b one-pot detection assay (Rcod) for Bordetella pertussis detection without additional amplification product transfer steps. The time from sample processing to response time was less than 30 min using nucleic acid extraction-free method, and the sensitivity reached 0.2 copies/μL. In this system, Alicyclobacillus acidoterrestris Cas12b protein (AacCas12b) exhibited strong and specific trans-cleavage activity at a constant temperature of 37 °C, while the cis-cleavage activity was weak. This characteristic reduces the interference of AacCas12b with nucleic acids in the system. Compared with real-time PCR, our Rcod system detected B. pertussis in 221 clinical samples with a sensitivity and specificity of 97.96 % and 99.19 %, respectively, with nucleic acid extraction-free method. The rapid, sensitive and specific Rcod system provides ideas for the establishment of CRISPR-based one-step nucleic acid detection and may aid the development of reliable point-of-care nucleic acid tests.
Pertussis is an acute respiratory infection caused by B. pertussis that is highly contagious and potentially fatal, and early diagnosis is essential for the treatment of whooping cough. In this study, we found that AacCas12b has high and strongly specific trans-cleavage activity at lower temperatures. A RAA-CRISPR/Cas12b one-step detection platform (Rcod) without interference with amplification was developed. In addition, the combination of Rcod and nucleic acid extraction-free method can quickly and accurately detect the qualitative detection of B. pertussis, and the detection results are visualized, which makes the pathogen nucleic acid detection and analysis process simpler, and provides a new method for the rapid clinical diagnosis of B. pertussis.
Plasmonic materials and manufacturing methods for rapid and sustainable thermal cycler for PCR
2023, Materials Today AdvancesMultiple outbreaks of fatal infectious diseases throughout history have intensified the need for early diagnostic methods to efficiently control their spread. Polymerase chain reaction (PCR)-based diagnosis is a sensitive, accurate, and effective method for detecting infections. However, conventional PCR-based diagnosis is slow and consumes large amounts of energy, primarily because of bulky, power-consuming thermal cyclers. Herein, we review recently published PCR-based diagnostic methods, in which plasmonic light-to-heat conversion-based thermal cyclers replace conventional ones. First, we explain the structures of recently developed rapid plasmonic-based thermal cyclers and review the various materials used. Next, we review the fabrication methods used in recent developments in rapid plasmonic thermal cyclers. Then, we discuss sustainable methods that have been and can be implemented to develop a rapid plasmonic thermal cycler. With this review, the requirements for developing a plasmonic-based sustainable PCR with high speed, accuracy, and sensitivity can be understood to contain future pandemics.
ITS alchemy: On the use of ITS as a DNA marker in fungal ecology
2023, Fungal EcologyHigh throughput sequencing of PCR amplicons derived from environmental DNA (aka DNA metabarcoding) has become an integral part of fungal ecology, enabling in-depth characterization of fungal communities. In most cases, the rDNA Internal Transcribed Spacer (ITS) region, which has a long history as a target in fungal systematics, is used as a DNA barcode marker. Despite improvements in sequencing techniques and bioinformatics approaches, there are inherent limitations associated with the use of a single-locus DNA marker that are often ignored. In this text, I discuss both inherent biological and methodological limitations associated with the use of the ITS marker. For example, proper species delimitation is often not possible with a single marker, and a significant DNA barcoding gap (i.e. interspecific divergence) is often missing between sister taxa in ITS. Further, we can rarely be fully confident about the assigned species-level taxonomy based on available reference sequences. In addition to the inherent limitations, an extra layer of complexity and variation is blended into DNA metabarcoding data due to PCR and sequencing errors that may look similar to natural molecular variation. The bioinformatics processing of ITS amplicons must take into account both the basic properties of the ITS region, as well as the generated errors and biases. In this regard, we cannot adopt approaches and settings from other markers, such as 16S and 18S, blindly. For example, due to intraspecific variability in the ITS region, and sometimes intragenomic variability, ITS sequences must be clustered to approach species level resolution in community studies. Therefore, I argue that the concept of amplicon sequence variants (ASVs) is not applicable. Although the ITS region is by far the best option as a general DNA (meta)barcoding marker for fungi, this contribution is meant to remind against a naive or simplistic use of the ITS region, and for stimulating further discussions.
Next-generation diagnostic test for dengue virus detection using an ultrafast plasmonic colorimetric RT-PCR strategy
2023, Analytica Chimica ActaThe current global COVID-19 pandemic once again highlighted the urgent need for a simple, cost-effective, and sensitive diagnostic platform that can be rapidly developed for distribution and easy access in resource-limited areas. Here, we present a simple and low-cost plasmonic photothermal (PPT)-reverse transcription-colorimetric polymerase chain reaction (RTcPCR) for molecular diagnosis of dengue virus (DENV) infection. The assay can be completed within 54 min with an estimated detection limit of 1.6 copies/μL of viral nucleic acid. The analytical sensitivity and specificity of PPT-RTcPCR were comparable to that of the reference RT-qPCR assay. Moreover, the clinical performance of PPT-RTcPCR was evaluated and validated using 158 plasma samples collected from patients suspected of dengue infection. The results showed a diagnostic agreement of 97.5% compared to the reference RT-qPCR and demonstrated a clinical sensitivity and specificity of 97.0% and 100%, respectively. The simplicity and reliability of our PPT-RTcPCR strategy suggest it can provide a foundation for developing a field-deployable diagnostic assay for dengue and other infectious diseases.
Performance evaluation of QuantStudio 1 plus real-time PCR instrument for clinical laboratory analysis: A proof-of-concept study
2023, Practical Laboratory MedicineThe real-time PCR system is one of the most powerful research tools available in the life sciences field. The aim of this study was to preliminarily evaluate the analytical performance of QuantStudio 1 Plus real-time PCR system (QS 1 plus) for clinical procedures.
The consistency of QS 1 plus with the reference system in terms of various clinical procedures was evaluated. For qualitative data, the Kappa test was used to analyze the agreement of the results. For the quantitative data, Passing-Bablok regression analysis and Bland-Altman plot analysis were used to assess the concordance between QS 1 plus and the reference instrument.
Passing-Bablok regression showed an excellent agreement between the QS 1 plus and LC 480 systems for HBV DNA quantification (y = 0.928 + 0.970x), whereas Bland-Altman plot analysis showed very small mean deviations between the two systems. The QS 1 plus yielded perfectly consistent results with the reference instrument for methylenetetrahydrofolate reductase (MTHFR) C677T melting curve genotyping analysis, MTHFR C677T genotyping analysis, Norovirus RNA negative/positive analysis, influenza B virus (Flu B) RNA negative/positive analysis, Mycobacterium tuberculosis (MTB) DNA negative/positive analysis, Human Papillomavirus (HPV) genotyping analysis, epidermal growth factor receptor (EGFR) gene mutation analysis. Both the relative quantitative analysis and the relative quantitative analysis (standard curve) confirmed the satisfactory concordance between the QS 1 plus instrument and the ABI 7500 instrument by Passing-Bablok regression analysis (y = 0.180 + 0.817x and y = 0.012 + 1.000x, respectively) and Bland-Altman plot analysis.
Our research has proven that QS 1 plus is adaptable to most test procedures in the clinical laboratory. This may provide the basis for its further application.
PCR inhibitors and facilitators – Their role in forensic DNA analysis
2023, Forensic Science InternationalSince its inception, DNA typing technology has been practiced as a robust tool in criminal investigations. Experts usually utilize STR profiles to identify and individualize the suspect. However, mtDNA and Y STR analyses are also considered in some sample-limiting conditions. Based on DNA profiles thus generated, forensic scientists often opine the results as Inclusion, exclusion, and inconclusive. Inclusion and exclusion were defined as concordant results; the inconclusive opinions create problems in conferring justice in a trial- since nothing concrete can be interpreted from the profile generated. The presence of inhibitor molecules in the sample is the primary factor behind these indefinite results. Recently, researchers have been emphasizing studying the sources of PCR inhibitors and their mechanism of inhibition. Furthermore, several mitigation strategies- to facilitate the DNA amplification reaction -have now found their place in the routine DNA typing assays with compromised biological samples. The present review paper attempts to provide a comprehensive review of PCR inhibitors, their source, mechanism of inhibition, and ways to mitigate their effect using PCR facilitators.