scholarly journals Multiplexed detection of bacterial nucleic acids using Cas13 in droplet microarrays

2021 ◽  
Author(s):  
Sri Gowtham Thakku ◽  
Cheri M Ackerman ◽  
Cameron Myhrvold ◽  
Roby P Bhattacharyya ◽  
Jonathan Livny ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Sensitivity And Specificity ◽  
Health Management ◽  
Point Of Care ◽  
Bacterial Species ◽  
Bacterial Pathogen ◽  
Antibiotic Resistance Genes ◽  
Detection Methods ◽  
Nucleic Acid Detection ◽  
Multiplexed Detection

Rapid and accurate diagnosis of infections is fundamental to individual patient care and public health management. Nucleic acid detection methods are critical to this effort, but are limited either in the breadth of pathogens targeted or by the expertise and infrastructure required. We present here a high-throughput system that enables rapid identification of bacterial pathogens, bCARMEN, which utilizes: (1) modular CRISPR-Cas13-based nucleic acid detection with enhanced sensitivity and specificity; and (2) a droplet microfluidic system that enables thousands of simultaneous, spatially multiplexed detection reactions at nanoliter volumes; and (3) a novel pre-amplification strategy that further enhances sensitivity and specificity. We demonstrate bCARMEN is capable of detecting and discriminating 52 clinically relevant bacterial species and several key antibiotic resistance genes. We further develop a proof of principle system for use with stabilized reagents and a simple workflow with optical readout using a cell phone camera, opening up the possibility of a rapid point-of-care multiplexed bacterial pathogen identification and antibiotic susceptibility testing.

scholarly journals Ultrasensitive CRISPR-based diagnostic for field-applicable detection ofPlasmodiumspecies in symptomatic and asymptomatic malaria

2020 ◽  
Vol 117 (41) ◽  
pp. 25722-25731 ◽  
Author(s):  
Rose A. Lee ◽  
Helena De Puig ◽  
Peter Q. Nguyen ◽  
Nicolaas M. Angenent-Mari ◽  
Nina M. Donghia ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Sensitivity And Specificity ◽  
Parasite Density ◽  
Point Of Care ◽  
High Sensitivity ◽  
World Health ◽  
Ultrasensitive Detection ◽  
Asymptomatic Malaria ◽  
Radical Cure ◽  
Asymptomatic Carriers

Asymptomatic carriers ofPlasmodiumparasites hamper malaria control and eradication. Achieving malaria eradication requires ultrasensitive diagnostics for low parasite density infections (<100 parasites per microliter blood) that work in resource-limited settings (RLS). Sensitive point-of-care diagnostics are also lacking for nonfalciparum malaria, which is characterized by lower density infections and may require additional therapy for radical cure. Molecular methods, such as PCR, have high sensitivity and specificity, but remain high-complexity technologies impractical for RLS. Here we describe a CRISPR-based diagnostic for ultrasensitive detection and differentiation ofPlasmodium falciparum,Plasmodium vivax,Plasmodium ovale, andPlasmodium malariae, using the nucleic acid detection platform SHERLOCK (specific high-sensitivity enzymatic reporter unlocking). We present a streamlined, field-applicable, diagnostic comprised of a 10-min SHERLOCK parasite rapid extraction protocol, followed by SHERLOCK for 60 min forPlasmodiumspecies-specific detection via fluorescent or lateral flow strip readout. We optimized one-pot, lyophilized, isothermal assays with a simplified sample preparation method independent of nucleic acid extraction, and showed that these assays are capable of detection below two parasites per microliter blood, a limit of detection suggested by the World Health Organization. OurP. falciparumandP. vivaxassays exhibited 100% sensitivity and specificity on clinical samples (5P. falciparumand 10P. vivaxsamples). This work establishes a field-applicable diagnostic for ultrasensitive detection of asymptomatic carriers as well as a rapid point-of-care clinical diagnostic for nonfalciparum malaria species and low parasite densityP. falciparuminfections.

scholarly journals Development and detection of genetically modified crops

2020 ◽  
Vol 145 ◽  
pp. 01013
Author(s):  
Zhao Yu-jia ◽  
Fan Pei-lei ◽  
Liang Liang ◽  
Liu Yin-yin ◽  
Zhao Hai-bo ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Genetically Modified ◽  
Genetically Modified Crops ◽  
Detection Methods ◽  
Social Benefits ◽  
Nucleic Acid Detection ◽  
Genetically Modified Crop ◽  
The World ◽  
The Difference

Genetically modified crops (GMCs) have been known for the excellent qualities. The commercializing of GMCs has taken great economic and social benefits. However, the bio-security of GMCs was still an issue. To solve this problem, countries around the world were constantly strengthening regulations on planting, processing and detecting of GMCs. This paper reviewed the development of commercialization and detection of GMCs. The difference between protein and nucleic acid detection methods of genetically modified crop was further discussed. This paper will provide new insights for the application of genetically modified crops.

scholarly journals Quality Management for Point-Of-Care Testing of Pathogen Nucleic Acids: Chinese Expert Consensus

2021 ◽  
Vol 11 ◽  
Author(s):  
Xi Mo ◽  
Xueliang Wang ◽  
Zhaoqin Zhu ◽  
Yuetian Yu ◽  
Dong Chang ◽  
...  
Keyword(s):  
Public Health ◽  
Nucleic Acid ◽  
Quality Management ◽  
Clinical Laboratory ◽  
Point Of Care ◽  
Conventional Polymerase Chain Reaction ◽  
Laboratory Medicine ◽  
Expert Consensus ◽  
Nucleic Acid Detection ◽  
Point Of Care Testing

COVID-19 continues to circulate globally in 2021, while under the precise policy implementation of China’s public health system, the epidemic was quickly controlled, and society and the economy have recovered. During the pandemic response, nucleic acid detection of SARS-CoV-2 has played an indispensable role in the first line of defence. In the cases of emergency operations or patients presenting at fever clinics, nucleic acid detection is required to be performed and reported quickly. Therefore, nucleic acid point-of-care testing (POCT) technology for SARS-CoV-2 identification has emerged, and has been widely carried out at all levels of medical institutions. SARS-CoV-2 POCT has served as a complementary test to conventional polymerase chain reaction (PCR) batch tests, thus forming an experimental diagnosis platform that not only guarantees medical safety but also improves quality services. However, in view of the complexity of molecular diagnosis and the biosafety requirements involved, pathogen nucleic acid POCT is different from traditional blood-based physical and chemical index detection. No guidelines currently exist for POCT quality management, and there have been inconsistencies documented in practical operation. Therefore, Shanghai Society of Molecular Diagnostics, Shanghai Society of Laboratory Medicine, Clinical Microbiology Division of Shanghai Society of Microbiology and Shanghai Center for Clinical Laboratory have cooperated with experts in laboratory medicine to generate the present expert consensus. Based on the current spectrum of major infectious diseases in China, the whole-process operation management of pathogen POCT, including its application scenarios, biosafety management, personnel qualification, performance verification, quality control, and result reporting, are described here. This expert consensus will aid in promoting the rational application and robust development of this technology in public health defence and hospital infection management.

scholarly journals A Chemical-Enhanced System for CRISPR-Based Nucleic Acid Detection

2021 ◽  
Author(s):  
Zihan Li ◽  
Wenchang Zhao ◽  
Shixin Ma ◽  
Zexu Li ◽  
Yingjia Yao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Detection System ◽  
Point Of Care ◽  
Detection Sensitivity ◽  
Nucleic Acid Detection ◽  
Chemical Additives ◽  
Lateral Flow Strip ◽  
Viral Pathogens ◽  
Detection Systems ◽  
System Robustness

The CRISPR-based nucleic acid detection systems such as SHERLOCK, DETECTR and HOLMES have shown great potential for point-of-care testing of viral pathogens, especially in the context of COVID-19 pandemic. Here we optimize several key parameters of reaction chemistry and develop a Chemical Enhanced CRISPR Detection system for nucleic acid (termed CECRID). For the Cas12a/Cas13a-based signal detection phase, we determine buffer conditions and substrate range for optimal detection performance. By comparing several chemical additives, we find that addition of L-proline can secure or enhance Cas12a/Cas13a detection capability. For isothermal amplification phase with typical LAMP and RPA methods, inclusion of L-proline can also enhance specific target amplification as determined by CRISPR detection. Using SARS-CoV-2 pseudovirus, we demonstrate CECRID has enhanced detection sensitivity over chemical additive-null method with either fluorescence or lateral flow strip readout. Thus, CECRID provides an improved detection power and system robustness towards practical application of CRISPR-based diagnostics.

scholarly journals CRISPR-Assisted DNA Detection, a novel dCas9-based DNA detection technique

2020 ◽  
Author(s):  
Xinhui Xu ◽  
Tao Luo ◽  
Jinliang Gao ◽  
Na Lin ◽  
Weiwei Li ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Room Temperature ◽  
Dna Detection ◽  
Point Of Care ◽  
Hpv Infection ◽  
Solid Support ◽  
Human Papillomaviruses ◽  
Nucleic Acid Detection ◽  
Detection Techniques ◽  
Signal Readout

AbstractNucleic acid detection techniques are always critical to diagnosis, especially in the background of the present COVID-19 pandemic. The simple and rapid detection techniques with high sensitivity and specificity are always urgently needed. However, the current nucleic acid detection techniques are still limited the traditional amplification and hybridization. To overcome the limitation, we here develop a CRISPR/Cas9-assisted DNA detection (CADD). In this detection, DNA sample is incubated with a pair of capture sgRNAs (sgRNAa and sgRNAb) specific to a target DNA, dCas9, a signal readout-related probe, and an oligo-coated solid support beads or microplate at room temperature for 15 min. During this incubation, the dCas9-sgRNA-DNA complex is formed and captured on solid support by the capture sequence of sgRNAa and the signal readout-related probe is captured by the capture sequence of sgRNAb. Finally the detection result is reported by a fluorescent or colorimetric signal readout. This detection was verified by detecting DNA of bacteria, cancer cell and virus. Especially, by designing a set of sgRNAs specific to 15 high-risk human papillomaviruses (HPVs), the HPV infection in 64 clinical cervical samples were successfully detected by the method. All detections can be finished in 30 minutes at room temperature. This detection holds promise for rapid on-the-spot detection or point-of-care testing (POCT).

Pyrococcus furiosus Argonaute-mediated nucleic acid detection

2019 ◽  
Vol 55 (88) ◽  
pp. 13219-13222 ◽  
Author(s):  
Ruyi He ◽  
Longyu Wang ◽  
Fei Wang ◽  
Wenqiang Li ◽  
Yang Liu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Pyrococcus Furiosus ◽  
Nucleic Acid Detection ◽  
Single Nucleotide ◽  
Multiplexed Detection

PfAgo-mediated Nucleic acid Detection (PAND) distinguishes single-nucleotide mutants and accomplishes multiplexed detection by a second round of cleavage.

scholarly journals Conventional and Molecular Detection Methods of the Opportunistic Bacterial Pathogen Campylobacter concisus

2021 ◽  
Author(s):  
Mohsina Huq ◽  
Taghrid Istivan
Keyword(s):  
Molecular Typing ◽  
Bacterial Species ◽  
Acute Infection ◽  
Bacterial Pathogen ◽  
Clinical Importance ◽  
Detection Methods ◽  
Campylobacter Concisus ◽  
Bowel Diseases ◽  
Typing Methods ◽  
Inflammatory Bowel

Campylobacter concisus is an emerging pathogen that causes gastroenteritis and is a suspected cause of inflammatory bowel diseases. Its importance is enhanced by the chronic sequela that results from acute infection. This bacterium has been under-diagnosed in intestinal infectious diseases, and its clinical importance has not been determined yet. In order to establish the implication of this emerging bacterial species in human gastroenteritis and other infections, different approaches and procedure have been performed, where molecular typing methods have played a central role. The chapter provides a comprehensive past and recent updates on the detection of C. concisus by biochemical and molecular methods.

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