Pre-amplification Step Research Articles

The Medawar Prize Acceptance Speech 2022.

The Medawar Prize Acceptance Speech 2022.

Multiple crRNAs-assisted CRISPR/Cas12a assay targeting cytochrome b gene for amplification-free detection of meat adulteration

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems have been widely applied in nucleic acid analysis for the high specificity. Coupled with pre-amplification steps, the sensitivity of CRISPR-based detection is greatly improved. However, an extra pre-amplification step not only complicates the detection procedures but may also cause aerosol contaminations in the process of transferring amplified solution into CRISPR system. In this study, we demonstrate that combination of multiple crRNAs in CRISPR/Cas12a system can enhance the detection sensitivity. Based on it, we establish a multiple crRNAs enhanced CRISPR (meCRISPR) method and apply it to meat adulteration identification. Take cytochrome b (Cyt b) gene as a target, meCRISPR method can directly detect as low as 1.13 ng/μL extracted pork DNA and 5% (w/w) pork contamination in pork and beef meat mixtures. There is no cross-reaction with extracted chicken, beef, duck and fish DNA. meCRISPR reaction is incubated at an isothermal temperature, and the detection process can be completed in a designed portable apparatus with a heat block, a light emitting diode and filters. For the simplicity, specificity and sufficient sensitivity of meCRISPR method, it will have great prospects in species identification, food adulteration, and genetically modified food detection.

Methodological considerations for circulating long noncoding RNA quantification.

In the past decade, significant resources have been invested in long noncoding RNA (lncRNA) research. Despite the knowledge available, we are far from incorporation of lncRNA into clinical practice. Here, we emphasize the technical challenges in the field, hoping to provoke a response leading to new consensus and guidelines.

Recent Advances in CRISPR-Based Biosensors for Point-of-Care Pathogen Detection.

Infectious pathogens are pressing concerns due to their heavy toll on global health and socioeconomic infrastructure. Rapid, sensitive, and specific pathogen detection methods are needed more than ever to control disease spreading. The fast evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics (CRISPR-Dx) has opened a new horizon in the field of molecular diagnostics. This review highlights recent efforts in configuring CRISPR technology as an efficient diagnostic tool for pathogen detection. It starts with a brief introduction of different CRISPR-Cas effectors and their working principles for disease diagnosis. It then focuses on the evolution of laboratory-based CRISPR technology toward a potential point-of-care test, including the development of new signaling mechanisms, elimination of preamplification and sample pretreatment steps, and miniaturization of CRISPR reactions on digital assay chips and lateral flow devices. In addition, promising examples of CRISPR-Dx for pathogen detection in various real samples, such as blood, saliva, nasal swab, plant, and food samples, are highlighted. Finally, the challenges and perspectives of future development of CRISPR-Dx for infectious disease monitoring are discussed.

Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters.

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.

Diagnostics of Infections Produced by the Plant Viruses TMV, TEV, and PVX with CRISPR-Cas12 and CRISPR-Cas13.

Viral infections in plants threaten food security. Thus, simple and effective methods for virus detection are required to adopt early measures that can prevent virus spread. However, current methods based on the amplification of the viral genome by polymerase chain reaction (PCR) require laboratory conditions. Here, we exploited the CRISPR-Cas12a and CRISPR-Cas13a/d systems to detect three RNA viruses, namely, Tobacco mosaic virus, Tobacco etch virus, and Potato virus X, in Nicotiana benthamiana plants. We applied the CRISPR-Cas12a system to detect viral DNA amplicons generated by PCR or isothermal amplification, and we also performed a multiplexed detection in plants with mixed infections. In addition, we adapted the detection system to bypass the costly RNA purification step and to get a visible readout with lateral flow strips. Finally, we applied the CRISPR-Cas13a/d system to directly detect viral RNA, thereby avoiding the necessity of a preamplification step and obtaining a readout that scales with the viral load. These approaches allow for the performance of viral diagnostics within half an hour of leaf harvest and are hence potentially relevant for field-deployable applications.

Abstract 535: Development of a liquid biopsy assay to longitudinally monitor changes in mutations’ allelic frequency in response to treatment with KB-0742

Abstract Liquid biopsies analyzing cell-free DNA (cfDNA) released by tumors are emerging as a powerful noninvasive sampling approach. As opposed to single-site solid tumor biopsy, liquid biopsies allow for better representation of the tumor mutational landscape. In the clinic, liquid biopsies are utilized for early detection of disease, to help inform treatment decisions, to monitor for disease recurrence, and to identify potential resistance mechanisms. Here we describe our approach to developing a liquid biopsy assay that will allow us to monitor, in real time, longitudinal changes in mutations’ allelic frequency in patients with solid tumors. Our approach combines two powerful technologies: next-generation sequencing, that enables the identification of a broad array of mutations, and droplet digital PCR (ddPCR) that provides speed and a cost-effective advantage to monitor mutations in cfDNA obtained from patient plasma samples. In order to maximize the effectiveness of using liquid biopsy assays in our clinical trial, we prioritized developing an approach that enabled us to meet the following criteria: 1) generating a baseline and end-of-treatment mutational profile for each patient using either a tumor tissue sample or liquid biopsy to identify both driver and potential resistance mutation markers, 2) longitudinal sampling and real-time analysis over the course of treatment, 3) high assay sensitivity to detect low frequency alleles, 4) overall cost-effectiveness, and 5) ability to examine allelic frequency of multiple mutations of interest from limited input of cfDNA isolated from plasma. Assay development included custom quantification of cfDNA and optimization of a pre-amplification step, which amplified the material allowing us to analyze multiple mutations of interest while ensuring that the quantitative nature of the assay was not impacted. For each individual mutation, gBlocks™ and/or cell-line−derived cfDNA were utilized to optimize annealing temperature and time, cycle number, and input amount to attain an assay with a target 0.1% limit of detection. These customized mutation assays can potentially serve as an accelerated cost-effective surrogate approach, compared to imaging, to monitor in real time the response to KB-0742 (CDK9i) in the ongoing phase 1/2 clinical trial NCT04718675. Citation Format: Suha Naffar-Abu Amara, Christina Noe, Tressa Hood, Sara Paganini, Crystal MacKenzie, Melinda A. Day, Pavan Kumar. Development of a liquid biopsy assay to longitudinally monitor changes in mutations’ allelic frequency in response to treatment with KB-0742 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 535.

DigiMir Test: Establishing a Novel Pipeline for MiR-371a Quantification Using Droplet Digital PCR in Liquid Biopsies From Testicular Germ Cell Tumor Patients.

Testicular germ cell tumors (TGCTs) are the most common cancers in young-adult male patients aged between 15 and 39 years. Hsa-miR-371a-3p is currently the most reliable biomarker for diagnosis and monitoring of these patients non-invasively in liquid biopsies, and it is destined to be introduced in the clinic due to improved performance compared to the classical serum tumor markers available. Current studies have focused on real-time quantitative PCR (RT-qPCR) protocols for its determination; still, some challenges remain, since these protocols often require preamplification steps (costly and time-consuming), and report relative levels normalized to a housekeeping microRNA, not always performed the same way. Droplet digital PCR (ddPCR) shows the promise to overcome these challenges, skipping normalization and preamplifications, but has hardly been explored in the field of TGCTs. In this work, we provide a report of a ddPCR-based pipeline for the quantification of hsa-miR-371a-3p (the DigiMir pipeline) and compare it with two RT-qPCR protocols. A total of 107 plasma samples were investigated in the validation setting. The DigiMir pipeline detected TGCTs in a manner representative of tumor burden, with a sensitivity and specificity of 94% and 100%, respectively, outperforming the combined sensitivity of all three classical serum tumor markers (61.5%). Therefore, in this proof-of-concept investigation, we have shown that the DigiMir pipeline constitutes a new promising methodology to accurately report hsa-miR-371a-3p in the clinical setting.

Diagnostics of COVID-19 Based on CRISPR-Cas Coupled to Isothermal Amplification: A Comparative Analysis and Update.

The emergence of the COVID-19 pandemic prompted fast development of novel diagnostic methods of the etiologic virus SARS-CoV-2. Methods based on CRISPR–Cas systems have been particularly promising because they can achieve a similar sensitivity and specificity to the benchmark RT-qPCR, especially when coupled to an isothermal pre-amplification step. Furthermore, they have also solved inherent limitations of RT-qPCR that impede its decentralized use and deployment in the field, such as the need for expensive equipment, high cost per reaction, and delivery of results in hours, among others. In this review, we evaluate publicly available methods to detect SARS-CoV-2 that are based on CRISPR–Cas and isothermal amplification. We critically analyze the steps required to obtain a successful result from clinical samples and pinpoint key experimental conditions and parameters that could be optimized or modified to improve clinical and analytical outputs. The COVID outbreak has propelled intensive research in a short time, which is paving the way to develop effective and very promising CRISPR–Cas systems for the precise detection of SARS-CoV-2. This review could also serve as an introductory guide to new labs delving into this technology.

When Mosquito HV bites Biomark HD: An automated workflow for high-throughput qPCR.

Automation solutions can significantly improve sample processing efficiency and can be of particular help in core facility settings. Centralized core facilities are being established world-wide aimed at strengthening institutions' clinical and research enterprises and at addressing the need to process large volumes of samples on expensive cutting-edge technologies in a limited time. High-throughput qPCR profiling is a service offered by most genomics facilities. Several platforms have been developed to process large numbers of samples in a short time, including the Fluidigm Biomark HD, which has also been proved useful to increase the SARS-CoV-2 testing capacities. Several automation systems are currently available to miniaturize volumes and improve bioanalytical workflows, including the SPT Labtech Mosquito HV system. Here we have applied the Mosquito HV platform for the automation of the sample preparation of the Fluidigm gene expression workflow. We have successfully automated the pre-amplification and exonuclease cleanup steps with the aim of reducing manual error and sample processing time. We show consistency in the expression of reference genes when assessing pooled RNA control samples for the manual and automated workflows of Fluidigm gene expression profiling.

Rapid and sensitive detection of Ebola RNA in an unamplified sample based on CRISPR-Cas13a and DNA roller machine

A fast and simple Cas13a-based assay approach for direct detecting Ebola RNA in unamplified samples is reported. The procedure (named Cas-Roller) is comprised of a 10-min Cas13a-mediated cleavage protocol, followed by a DNA roller running for 30 min. This involves Cas13a collateral cleaving a suitably designed substrate in the presence of Ebola virus RNA sequence, and the cleavage product is used for DNA roller to amplify and generate fluorescent signals. After optimization of the conditions, the assay is able to achieve a limit of detection as low as 291 aM (∼175 copies RNA/μL) along with excellent anti-interfering performance in human serum and blood detection, which is ∼310-fold improved compared with the direct CRISPR assay. The entire workflow can be completed in ∼40 min at 37 °C without any pre-amplification, transcription, or centrifugation steps, thus avoiding the generation of false-negative or positive results. In addition, the downstream roller reaction is independent of the target sequence, this method can be applied to detect any other RNA by merely redesigning the hybridization regions of the crRNA. Overall, this strategy gives a new idea for the construction of simple and accurate Cas13a-based assays for the direct detection of RNA.

Design and implementation of multiplexed amplicon sequencing panels to serve genomic epidemiology of infectious disease: A malaria case study.

Multiplexed PCR amplicon sequencing (AmpSeq) is an increasingly popular application for cost-effective monitoring of threatened species and managed wildlife populations, and shows strong potential for the genomic epidemiology of infectious disease. AmpSeq data from infectious microbes can inform disease control in multiple ways, such as by measuring drug resistance marker prevalence, distinguishing imported from local cases, and determining the effectiveness of therapeutics. We describe the design and comparative evaluation of two new AmpSeq assays for Plasmodium falciparum malaria parasites: a four-locus panel ("4CAST") composed of highly diverse antigens, and a 129-locus panel ("AMPLseq") composed of drug resistance markers, highly diverse loci for inferring relatedness, and a locus to detect Plasmodium vivax co-infection. We explore the performance of each panel in various public health use cases with in silico simulations as well as empirical experiments. The 4CAST panel appears highly suitable for evaluating the number of distinct parasite strains within samples (complexity of infection), showing strong performance across a wide range of parasitaemia levels without a DNA pre-amplification step. For relatedness inference, the larger AMPLseq panel performs similarly to two existing panels of comparable size, despite differences in the data and approach used for designing each panel. Finally, we describe an R package (paneljudge) that facilitates the design and comparative evaluation of genetic panels for relatedness estimation, and we provide general guidance on the design and implementation of AmpSeq panels for the genomic epidemiology of infectious disease.

Rapid identification of tumor-reactive T-cell receptors by RNA preamplification-based single-cell sequencing

T-cell receptor (TCR)-transduced T (TCR-T) cell therapy has shown promising efficacy in the clinical treatment of malignant cancers. However, the populations covered by reported TCRs are still limited. Tumor infiltrating lymphocytes (TILs) are natural reservoirs of tumor-reactive T cells and TCRs. Approaches are required for the fast and cost-effective identification of tumor-reactive TCRs from TILs. The widely employed TCR identification approaches by the clonal expansion of TILs involve a TCR singularization process for the direct pairing of TCR Vα and the Vβ chain. However, the clonal expansion of T cells is well known to require extensive time and effort due to the involvement of T cell cultures. Several single-cell multiplexing PCR methods followed by Sanger sequencing have been developed, representing a cost-effective and fast approach for single-cell TCR identification. In this study, an RNA-based preamplification step was included in the single-cell TCR sequencing, which can reduce the multiplexing PCR amplification to one round. Moreover, the cDNA product of RNA preamplification is derived from the whole genome mRNA, instead of TCR mRNA only by multiplexing primers-based DNA preamplification, which is valuable for many other analyses (e.g., phenotypic analysis) of the tumor-reactive T cells that can be correlated with the identified TCRs. The feasibility for both single α chain and dual α chain TILs of this approach highlights its potential value as a rapid and cost-effective sequencing strategy for the development of TCR-T therapies for solid cancers.

Genomic analysis of human noroviruses using combined Illumina-Nanopore data.

Whole-genome sequence analysis of noroviruses is routinely performed by employing a metagenomic approach. While this methodology has several advantages, such as allowing for the examination of co-infection, it has some limitations, such as the requirement of high viral load to achieve full-length or near full-length genomic sequences. In this study, we used a pre-amplification step to obtain full-length genomic amplicons from 39 Canadian GII isolates, followed by deep sequencing on Illumina and Oxford Nanopore platforms. This approach significantly reduced the required viral titre to obtain full-genome coverage. Herein, we compared the coverage and sequences obtained by both platforms and provided an in-depth genomic analysis of the obtained sequences, including the presence of single-nucleotide variants and recombination events.

CRISPR-cas12a mediated SERS lateral flow assay for amplification-free detection of double-stranded DNA and single-base mutation

Lateral flow assay (LFA) is user-friendly diagnostic tools but suffering limitations in poorer sensitivities and specificities especially for double-stranded DNA and single-base mutation quantification. Here, to improve the sensitivity and specificity for the LFA based nucleic acid detection, CRISPR-Cas12a mediated Surface enhanced Raman scattering (SERS) LFA was developed. By combination of ultra-sensitive SERS tags and target-specific signal amplification ability of CRISPR-Cas12a, HIV-1 dsDNA can be directly quantified with a LOD of 0.3 fM without any pre-amplification steps, which is almost 4 orders of magnitude lower than that of traditional colorimetric LFA methods. The whole detection process can be finished less than 1 h. Moreover, based on the target specificity of Cas12a, HIV-1 single-based drug resistance mutation (M184V) can be recognized as low as 0.01%. The HIV-1 dsDNA can also be successfully detected in serum samples with good comparable of that in buffer setting. Therefore, the simple and inexpensive paper-based CRISPR-SERS strip has great potential for point-of-care testing (POCT) of nucleic acid targets especially in resource-poor or non-laboratory environments.

Argonaute with stepwise endonuclease activity promotes specific and multiplex nucleic acid detection

Argonaute proteins (Agos) from thermophiles function as endonucleases via guide-target base-pairing cleavage for host defense. Since guides play a key role in regulating the catalytic specificity of Agos, elucidating its underlying molecular mechanisms would promote the application of Agos in the medical sciences. Here, we reveal that an Ago from Pyrococcus furiosus (PfAgo) showed a stepwise endonuclease activity, which was demonstrated through a double-stranded DNA cleavage directed by a single guide DNA (gDNA) rather than a canonical pair of gDNAs. We validated that the cleavage products with 5'-phosphorylated ends can be used as a new guide to induce a new round of cleavage. Based on the reprogrammable capacity of Ago’s stepwise activity, we established a rapid and specific platform for unambiguous multiplex gene detection, termed Renewed-gDNA Assisted DNA cleavage by Argonaute (RADAR). Combined with a pre-amplification step, RADAR achieved sensitivity at the femtomolar level and specificity with at least a di-nucleotide resolution. Furthermore, RADAR simultaneously discriminated among multiple target sequences simply by corresponding multiple guides. We successfully distinguished four human papillomavirus serotypes from patient samples in a single reaction. Our technique, based on the unique properties of Ago, provides a versatile and sensitive method for molecular diagnosis.

A Standardized Protocol for Donor-Derived Cell-Free DNA Quantification in the Diagnosis of Allograft Injury

Purpose There is an increasing interest in the use of donor-derived cfDNA (dd-cfDNA) in transplantation for non-invasive diagnosis of allograft injury. We hereby describe a non-commercial routine procedure for dd-cfDNA detection and quantification using digital droplet PCR and target-specific preamplification of a SNP-panel of 35 SNP-assays. The method is based on the difference in genotype between recipients and donors. After establishing our protocol, it has been used in a prospective study following recipients for 12 months after heart transplantation (HTx), and we here briefly describe our protocol. Methods A total of 728 samples from 67 HTx patients have been retrieved and analyzed. The recipients were genotyped preoperatively with respect to the SNP-panel. Upon transplantation, the donor genotype was interrogated with respect to the homozygous alleles found in the recipient. After HTx, blood samples were collected in cell free DNA collection tubes and delivered within the same day to the hospital laboratory. Plasma was separated by centrifugation at 2000g and 16000g, respectively. Isolated plasma was stored at -80C or used immediately for cfDNA extraction. Quality controls were performed by the 4200 Tape Station. The same primers were used for preamplification and downstream PCR. Non-template controls were included in all preamplification steps. The amount of dd-cfDNA was calculated as a fraction of the total cfDNA. Results The variation of extracted cfDNA yield was 10-130 ng/ml plasma. The 50-800 bp sized cfDNA constitutes 90% of the extracted samples, indicating a low background. An average of 2-5 informative SNPS assays for each transplant recipient were used for dd-cfDNA quantification with a detection rate down to 0.01% dd-cfDNA. Elevation of dd-cfDNA was seen not only in conjunction with cellular rejection but also during infection and in episodes of heart failure Conclusion Samples from HTx recipients demonstrated low levels of dd-cfDNA in consecutive samples during optimal treatment in non-eventful cases. Elevation of dd-cfDNA was seen not only during rejection but also in other conditions. This non-invasive method is relatively cheap, enables large sample yields by preamplification, has a fast turn-around time (48 hours) and is thus useful for clinical monitoring of HTx patients. However, it needs to be carefully related to clinical events.

Application of microfluidic high-throughput quantitative PCR systems for the monitoring and surveillance of aquatic species

Quantitative PCR (qPCR) has been increasingly used for the detection of target organisms in environmental DNA (eDNA) studies, and this is thanks to high sensitivity and ability to quantify DNA targets copy number. However, prior to their implementation, qPCR species-specific assays must be developed and validated and, when implemented, they are limited to relatively low number of targets that can be screened as a multiplex or in parallel. Thanks to recent technological advances, several qPCR-based platforms have become available to increase the throughput capability of qPCR systems as well as lowering time of execution and costs associated to sample processing. The present study describes the use of a microfluidic high-throughput qPCR/dPCR system (Biomark HD, Fluidigm) for the screening of species of ecologic and economic importance in bulk plankton environmental samples from marine coastal areas around the Irish coast. Data was generated using the configuration enabling the highest throughput (in terms of data points) of the system, including Integrated Fluidic Control (IFC) units capable of producing 96 x 96 sample/assay combinations in each run (9,216 individual qPCR tests in a single run). Thanks to such a capability, it was possible to execute the following three main development and implementation phases in a relatively short period of time (weeks as opposed to months/years): (i) development of a panel of species-specific assays targeting a range of crustacean and bivalve species; (ii) assessment of Limits of Detection (LOD), Limits of Quantification (LOQ), and enzymatic inhibition control for selected assays; and (iii) screening of environmental time-series samples (n = 242) obtained from a citizen-like sampling effort that involved a range of stakeholders and locations throughout the Irish marine coastal territory between 2019 and 2020. During the assay-development phase, the IFC system configuration (whereby all assays are tested in parallel against all samples) enabled the rapid screening of species-specific assays against a wide range of (genomic DNA of) non-target organisms, hence enabling for rapid specificity testing. LOD/LOQ experiments showed high levels of sensitivity and thanks to the large number of assays that could be accommodated in a single run, it was possible to include up to four distinct Internal Positive Controls (IPCs) at different concentrations in each run (hence controlling for potential inhibition at different target concentration levels). The inclusion of inhibitor-removal reagents in a pre-amplification step as well as the dilution factor of conducting reactions in small volumes (6.7 nL reaction volumes, hence comparable to a “digital PCR” effect) proved to be an effective strategy to reduce the effect of inhibitors in control experiments (humic acid and EDTA), as well as in actual environmental samples from a range of marine environments. Combining such a high-throughput screening platform with a nation-wide citizen science-like sampling programme enabled the acquisition of large datasets that are being used to monitor occurrence and (spawning) activity of important species that are of conservation concern, commercial value, or non-indigenous and invasive to Irish waters. The Biomark HD system provides a remarkable flexibility to modify existing and/or incorporate new assays because IFCs are customizable just prior to usage (i.e. are not pre-loaded or spotted with primers/probes), thus current work is focussing on increasing the number of species targeted in a single run, and (thanks to the quantitative nature of data) discriminating between different fractions of DNA in heterogeneous bulk samples (e.g. gametes and larvae vs intra- and extracellular eDNA). Thanks to low sample processing cost, assay flexibility and high-throughput capability, microfluidic qPCR platforms behold the potential to significantly advance biomonitoring of aquatic ecosystems.

Pre-amplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs.

IntroductionThe assessment of circulating miRNAs is challenging and still limited due to their low concentrations, small size and lack of reference values in human biological samples. Pre-amplification of complementary DNAs may facilitate reliable miRNA quantification. The aim of our study was to evaluate the efficacy of pre-amplification as a step to increase the sensitivity of qPCR analysis for five candidate circulating miRNAs presumably related to toxic metals and cigarette smoke exposure: miR-1537, miR-190b, miR-16, miR-21, and miR-146a.Materials and methodsCandidate miRNAs expression was analysed in plasma samples of 19 mother-newborn pairs. For isolation, transcription, pre-amplification and qPCR quantification kits and protocols by Qiagen (Hilden, Germany) were used. Paired t-test or Wilcoxon rank test were used to compare miRNAs expression levels with and without a pre-amplification step prior to qPCR, separately in maternal and cord plasma. Intraclass correlation (ICC) was calculated as an agreement measure between procedures for each miRNA.ResultsPre-amplification facilitated the detection of all assayed miRNAs with an overall cycle threshold (CT) improvement of 6.6 ± 0.89 (P < 0.05). Excellent ICCs (> 0.90) were found between data for preamplified and not preamplified miR-16, miR-21 and miR-146a. However, these correlations for low expressed miR-190b were moderate (0.79 in maternal; 0.61 in cord plasma) and poor for miR-1537 (0.49 in maternal; no correlation in cord plasma).ConclusionPre-amplification is a useful, necessary step in the analysis of miR-1537 and miR-190b as a reliable procedure facilitating extracellular miRNA expression detection in human plasma by real-time PCR quantification.

CRISPR-ENHANCE: An enhanced nucleic acid detection platform using Cas12a.

Rapid detection of nucleic acids is essential for clinical diagnosis of a wide range of infectious and non-infectious diseases. CRISPR-based diagnostic platforms are well-established for rapid and specific detection of nucleic acids but suffer from a low detection sensitivity without a target pre-amplification step. Our recently developed detection system, called CRISPR-ENHANCE, employs engineered crRNAs and optimized conditions to achieve a significantly higher sensitivity and enable femtomolar levels of nucleic acid detection even without target pre-amplification. Using the CRISPR-ENHANCE platform and following the methodology detailed in this paper, nucleic acid detection for low copy numbers can be achieved in less than an hour through either a fluorescence-based detection or a lateral flow assay. The step-by-step instructions provided, in addition to describing how to perform both assays, incorporate details on a LAMP/RT-LAMP-based target amplification step to enable detection of RNA, ssDNA and dsDNA. Furthermore, a protocol for in-house expression and purification of LbCas12a using CL7/lm7-based affinity chromatography, which has been used to achieve a high yield and purity of the enzyme in a single-step, is provided.