A versatile CRISPR Cas12a-based point-of-care biosensor enabling convenient glucometer readout for ultrasensitive detection of pathogen nucleic acids.

Pathogen nucleic acid detection is of great significance to control the spread of diseases caused by the viruses. Nevertheless, traditional methods for nucleic acid detection such as polymerase chain reaction (PCR) and oligonucleotide microarrays require bulky instruments, which restrain their point-of-care (POC) testing application. Here, we proposed a POC method enabling sensitive detection of pathogen nucleic acids by combining the clustered regularly interspaced short palindromic repeat (CRISPR) Cas12a-based assay and personal glucometer readout (PGM). The quantification of target pathogen DNA by PGM was achieved based on pathogen DNA activates Cas12a ssDNase to cleave magnetic bead-DNA-invertase reporter probe, and separated free invertase to catalyze hydrolysis of sucrose to glucose. Without using nucleic acid amplification technology, we demonstrated here dual signal amplifications based on Cas12a and invertase-mediated catalytic reactions, making it possible to sensitively detect HIV-related DNA or SARS-CoV-2 pseudovirus with the limits of detection of 11.0 fM and 50 copies/µL, respectively. This strategy also showed excellent selectivity as well as potential applicability for detection of HIV in human serum samples or of SARS-CoV-2 in saliva samples. Therefore, our CRISPR-PGM-based dual signal amplifications detection platform might offer a great promise in POC diagnosis of pathogen nucleic acids.

Probing the mutation independent interaction of DNA probes with SARS-CoV-2 variants through a combination of surface-enhanced Raman scattering and machine learning.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution has been characterized by the emergence of sets of mutations impacting the virus characteristics, such as transmissibility and antigenicity, presumably in response to the changing immune profile of the human population. The presence of mutations in the SARS-CoV-2 virus can potentially impact therapeutic and diagnostic test performances. We design and develop here a unique set of DNA probes i.e., antisense oligonucleotides (ASOs) which can interact with genetic sequences of the virus irrespective of its ongoing mutations. The probes, developed herein, target a specific segment of the nucleocapsid phosphoprotein (N) gene of SARS-CoV-2 with high binding efficiency which do not mutate among the known variants. Further probing into the interaction profile of the ASOs reveals that the ASO-RNA hybridization remains unaltered even for a hypothetical single point mutation at the target RNA site and diminished only in case of the hypothetical double or triple point mutations. The mechanism of interaction among the ASOs and SARS-CoV-2 RNA is then explored with a combination of surface-enhanced Raman scattering (SERS) and machine learning techniques. It has been observed that the technique, described herein, could efficiently discriminate between clinically positive and negative samples with ∼100% sensitivity and ∼90% specificity up to 63 copies/mL of SARS-CoV-2 RNA concentration. Thus, this study establishes N gene targeted ASOs as the fundamental machinery to efficiently detect all the current SARS-CoV-2 variants regardless of their mutations.

In-silico analysis of Covid-19 genome sequences of Indian origin: Impact of mutations in identification of SARS-Co-V2.

Covid-19 disease caused by SARS-CoV-2 is still being transmitted in developed and developing countries irrespective of healthcare setups. India with 1.3 billion people in the world is severely affected by Covid-19 with 11.3 million cases and 157 000 deaths so far. We have assessed the mismatches in WHO recommended rRT-PCR assays primer and probe binding regions against SARS-CoV-2 Indian genome sequences through in-silico bioinformatics analysis approach. Primers and probe sequences belonging to CN-CDC-ORF1ab from China and HKU-ORF1b from Hong Kong targeting ORF1ab gene while NIH-TH-N from Thailand, HKU-N from Hong Kong and US-CDCN-2 from USA targeting N genes displayed accurate matches (>98.3%) with the 2019 novel corona virus sequences from India. On the other hand, none of the genomic sequences displayed exact match with the primer/probe sequences belonging to Charité-ORF1b from Germany targeting ORF1ab gene. We think it will be worthwhile to release this information to the clinical and medical communities working in Indian Covid-19 frontline taskforce to tackle the recently emerging Covid-19 outbreaks as of March-2021.

Development and validation of a new triplex real-time quantitative reverse Transcriptase-PCR assay for the clinical detection of SARS-CoV-2.

To increase the repertoire of PCR based laboratory developed tests (LDTs) for the detection of SARS-CoV-2, we describe a new multiplex assay (SORP), targeting the SARS-CoV-2's, Spike and ORF8 genes. The widely used human RNaseP internal control was modified to specifically co-amplify the RNaseP mRNA. The SORP triplex assay was tested on a cohort (n = 372; POS = 144/NEG = 228) of nasopharyngeal flocked swab (NPFS) specimens, previously tested for the presence of SARS-CoV-2 using a PCR assay targeting E and RdRp genes. The overall sensitivity and specificity of the SORP assay was: 99.31% (95% CI: 96.22-99.98%), 100.0% (95% CI: 98.4-100%) respectively. The SORP assay could also detect a panel of variants of concern (VOC) from the B1.1.7 (UK) and B1.351 (SA) lineage. In summary, access to a repertoire of new SARS-CoV-2 LDT's would assist diagnostic laboratories in developing strategies to overcome some of the testing issues encountered during high-throughput SARS-CoV-2 testing.

Analysis and forecasting of global real time RT-PCR primers and probes for SARS-CoV-2.

Rapid tests for active SARS-CoV-2 infections rely on reverse transcription polymerase chain reaction (RT-PCR). RT-PCR uses reverse transcription of RNA into complementary DNA (cDNA) and amplification of specific DNA (primer and probe) targets using polymerase chain reaction (PCR). The technology makes rapid and specific identification of the virus possible based on sequence homology of nucleic acid sequence and is much faster than tissue culture or animal cell models. However the technique can lose sensitivity over time as the virus evolves and the target sequences diverge from the selective primer sequences. Different primer sequences have been adopted in different geographic regions. As we rely on these existing RT-PCR primers to track and manage the spread of the Coronavirus, it is imperative to understand how SARS-CoV-2 mutations, over time and geographically, diverge from existing primers used today. In this study, we analyze the performance of the SARS-CoV-2 primers in use today by measuring the number of mismatches between primer sequence and genome targets over time and spatially. We find that there is a growing number of mismatches, an increase by 2% per month, as well as a high specificity of virus based on geographic location.

[Rapid diagnostics of novel coronavirus infection by loop-mediated isothermal amplification].

This review presents the basic principles of application of the loop-mediated isothermal amplification (LAMP) reaction for the rapid diagnosis of coronavirus infection caused by SARS-CoV-2. The basic technical details of the method, and the most popular approaches of specific and non-specific detection of amplification products are briefly described. We also discuss the first published works on the use of the method for the detection of the nucleic acid of the SARS-CoV-2 virus, including those being developed in the Russian Federation. For commercially available and published LAMP-based assays, the main analytical characteristics of the tests are listed, which are often comparable to those based on the method of reverse transcription polymerase chain reaction (RT-PCR), and in some cases are even superior. The advantages and limitations of this promising methodology in comparison to other methods of molecular diagnostics, primarily RT-PCR, are discussed, as well as the prospects for the development of technology for the detection of other infectious agents.

A rapid diagnosis of SARS-CoV-2 using DNA hydrogel formation on microfluidic pores.

The coronavirus disease 2019 (COVID-19) pandemic has been a major public health challenge in 2020. Early diagnosis of COVID-19 is the most effective method to control disease spread and prevent further mortality. As such, a high-precision and rapid yet economic assay method is urgently required. Herein, we propose an innovative method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using isothermal amplification of nucleic acids on a mesh containing multiple microfluidic pores. Hybridization of pathogen DNA and immobilized probes forms a DNA hydrogel by rolling circle amplification and, consequently, blocks the pores to prevent fluid movement, as observed. Following optimization of several factors, including pore size, mesh location, and precision microfluidics, the limit of detection (LOD) for SARS-CoV-2 was determined to be 0.7 aM at 15-min incubation. These results indicate rapid, easy, and effective detection with a moderate-sized LOD of the target pathogen by remote point-of-care testing and without the requirement of any sophisticated device.

Establishment of method for dual simultaneous detection of PEDV and TGEV by combination of magnetic micro-particles and nanoparticles.

Transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) are the main pathogens causing viral diarrhea in pig, mixed infections of these two viruses are very common in intensive pig rearing. However, there is a lack of a method to simultaneously detect and distinguish PEDV and TGEV in preclinical levels. In this study, we aimed to establish a dual ultrasensitive nanoparticle DNA probe-based PCR assay (dual UNDP-PCR) based on functionalized magnetic bead enrichment and specific nano-technology amplification for simultaneous detection and distinguish diagnosis of PEDV and TGEV. The detection limit of dual UNDP-PCR for single or multiple infections of PEDV and TGEV is 25 copies/g, which is 400 times more sensitive than the currently known duplex RT-PCR, showing better specificity and sensitivity without cross-reaction with other viruses. For pre-clinical fecal samples, the dual UNDP-PCR showed a markedly higher positive detection rate (52.08%) than conventional duplex RT-PCR (13.21%), can rapidly and accurately identify targeted pathogens whenever simple virus infection or co-infection. In summary, this study provides a technique for detecting and distinguishing PEDV and TGEV in preclinical levels, which is high sensitivity, specificity, repeatability, low cost and broad application prospect.