scholarly journals Enhancing Colorimetric LAMP Amplification Speed and Sensitivity with Guanidine Chloride

2020 ◽  
Author(s):  
Yinhua Zhang ◽  
Guoping Ren ◽  
Jackson Buss ◽  
Andrew J. Barry ◽  
Gregory C. Patton ◽  
...  
Keyword(s):  
Guanidine Hydrochloride ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Simple Method ◽  
Diagnostic Assays ◽  
Dna And Rna ◽  
Target Dna ◽  
Versatile Technique ◽  
Guanidine Chloride ◽  
Primer Sets

AbstractLoop-mediated isothermal amplification (LAMP) is a versatile technique for detection of target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. The global SARS-CoV-2 pandemic has presented an urgent need for new and better diagnostic methods, with colorimetric LAMP utilized in numerous studies for SARS-CoV-2 detection. However, the sensitivity of colorimetric LAMP in early reports has been below that of the standard RT-qPCR tests, and we sought to improve performance. Here we report the use of guanidine hydrochloride and combined primer sets to increase speed and sensitivity in colorimetric LAMP, bringing this simple method up to the standards of sophisticated technique and enabling accurate and high-throughput diagnostics.

scholarly journals Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride

BioTechniques ◽  
2020 ◽  
Vol 69 (3) ◽  
pp. 178-185 ◽  
Author(s):  
Yinhua Zhang ◽  
Guoping Ren ◽  
Jackson Buss ◽  
Andrew J Barry ◽  
Gregory C Patton ◽  
...  
Keyword(s):  
Guanidine Hydrochloride ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Simple Method ◽  
Diagnostic Assays ◽  
Loop Mediated Isothermal Amplification ◽  
Dna And Rna ◽  
Versatile Technique ◽  
Primer Sets

Loop-mediated isothermal amplification (LAMP) is a versatile technique for detection of target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. The global SARS-CoV-2 pandemic has presented an urgent need for new and better diagnostic methods, with colorimetric LAMP utilized in numerous studies for SARS-CoV-2 detection. However, the sensitivity of colorimetric LAMP in early reports has been below that of the standard RT-qPCR tests, and we sought to improve performance. Here we report the use of guanidine hydrochloride and combined primer sets to increase speed and sensitivity in colorimetric LAMP, bringing this simple method up to the standards of sophisticated techniques and enabling accurate, high-throughput diagnostics.

Laboratory Methods for the Diagnosis of SARS-Cov-2

2022 ◽  
pp. 58-77
Author(s):  
Mohamed Echchakery ◽  
Samia Boussaa ◽  
Souad El Mouahid ◽  
Maryam Mountassir ◽  
Said El Hizazi ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Diagnostic Tests ◽  
Epidemiological Studies ◽  
Virus Antigen ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Laboratory Methods ◽  
Virus Surface ◽  
Diagnostic Assays ◽  
Human Endeavor

The coronavirus disease 2019 (COVID-19) which has become the pandemic par excellence of our time places pressure on various aspects of human endeavor and as such requires detailed study to better combat it. However, diagnostic tests were used to provide data on the incidence of COVID-19 and to assess the immune status of infected individuals. The objective of this chapter is to describe the diagnostic methods currently used to identify SARS-CoV-2 infection. Obtaining the first SARS-CoV-2 genome sequence was decisive for the development of molecular diagnostic assays that currently make it possible to diagnose and screen for the Sars-CoV-2 infection. Their uses depend on the target to be detected. Antigenic tests detect the presence of a virus antigen, which usually makes a proteinaceous part of the virus surface. The serology tests detect the presence of antibodies generated against SARS-CoV-2 and are also a relevant tool for epidemiological studies.

scholarly journals Electric-field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2

2020 ◽  
Author(s):  
Ashwin Ramachandran ◽  
Diego A. Huyke ◽  
Eesha Sharma ◽  
Malaya K. Sahoo ◽  
Niaz Banaei ◽  
...  
Keyword(s):  
Electric Field ◽  
Early Stage ◽  
Diagnostic Methods ◽  
Disease Spread ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Diagnostic Assays ◽  
Field Control

AbstractThe rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In a basic form of this assay, the CRISPR-Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex is activated when it highly specifically binds to target DNA, and the activated complex non-specifically cleaves single-stranded DNA reporter probes labeled with a fluorophore-quencher pair. We recently discovered that electric field gradients can be used to control and accelerate this CRISPR assay by co-focusing Cas12-gRNA, reporters, and target. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab sample. We here combine this ITP purification with loop-mediated isothermal amplification, and the ITP-enhanced CRISPR assay to achieve detection of SARS-CoV-2 RNA (from raw sample to result) in 30 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables a new modality for a suite of microfluidic CRISPR-based diagnostic assays.Significance statementRapid, early-stage screening is especially crucial during pandemics for early identification of infected patients and control of disease spread. CRISPR biology offers new methods for rapid and accurate pathogen detection. Despite their versatility and specificity, existing CRISPR-diagnostic methods suffer from the requirements of up-front nucleic acid extraction, large reagent volumes, and several manual steps—factors which prolong the process and impede use in low resource settings. We here combine on-chip electric-field control in combination with CRIPSR biology to directly address these limitations of current CRISPR-diagnostic methods. We apply our method to the rapid detection of SARS-CoV-2 RNA in clinical samples. Our method takes 30 min from raw sample to result, a significant improvement over existing diagnostic methods for COVID-19.

scholarly journals Electric field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2

2020 ◽  
Vol 117 (47) ◽  
pp. 29518-29525 ◽  
Author(s):  
Ashwin Ramachandran ◽  
Diego A. Huyke ◽  
Eesha Sharma ◽  
Malaya K. Sahoo ◽  
ChunHong Huang ◽  
...  
Keyword(s):  
Electric Field ◽  
Microfluidic Chip ◽  
Molecular Diagnostic ◽  
Nasopharyngeal Swab ◽  
Guide Rna ◽  
Diagnostic Assays ◽  
Field Gradients ◽  
Target Dna ◽  
Rapid Spread ◽  
Diagnostic Approaches

The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In one basic form of this assay, the CRISPR–Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex becomes activated only when it specifically binds to target DNA and cleaves it. The activated complex thereafter nonspecifically cleaves single-stranded DNA reporter probes labeled with a fluorophore−quencher pair. We discovered that electric field gradients can be used to control and accelerate this CRISPR assay by cofocusing Cas12–gRNA, reporters, and target within a microfluidic chip. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab samples. We here combine this ITP purification with loop-mediated isothermal amplification and the ITP-enhanced CRISPR assay to achieve detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA (from raw sample to result) in about 35 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables an alternate modality for a suite of microfluidic CRISPR-based diagnostic assays.

scholarly journals CRISPR as a Diagnostic Tool

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1162
Author(s):  
Seohyun Kim ◽  
Sangmin Ji ◽  
Hye Ran Koh
Keyword(s):  
Diagnostic Tool ◽  
Specific Binding ◽  
Diagnostic Methods ◽  
Specific Gene ◽  
Guide Rna ◽  
Engineering Technique ◽  
Target Dna ◽  
Target Binding ◽  
Genetic Engineering Technique ◽  
Disease Related Gene

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has recently gained growing attention as a diagnostic tool due to its capability of specific gene targeting. It consists of Cas enzymes and a guide RNA (gRNA) that can cleave the target DNA or RNA based on the sequence of the gRNA, making it an attractive genetic engineering technique. In addition to the target-specific binding and cleavage, the trans-cleavage activity was reported for some Cas proteins, including Cas12a and Cas13a, which is to cleave the surrounding single-stranded DNA or RNA upon the target binding of Cas-gRNA complex. All these activities of the CRISPR-Cas system are based on its target-specific binding, making it applied to develop diagnostic methods by detecting the disease-related gene as well as microRNAs and the genetic variations such as single nucleotide polymorphism and DNA methylation. Moreover, it can be applied to detect the non-nucleic acids target such as proteins. In this review, we cover the various CRISPR-based diagnostic methods by focusing on the activity of the CRISPR-Cas system and the form of the target. The CRISPR-based diagnostic methods without target amplification are also introduced briefly.

scholarly journals SARS-CoV-2 IgG Antibodies Seroprevalence and Sera Neutralizing Activity in MEXICO: A National Cross-Sectional Study during 2020

2021 ◽  
Vol 9 (4) ◽  
pp. 850
Author(s):  
José Esteban Muñoz-Medina ◽  
Concepción Grajales-Muñiz ◽  
Angel Gustavo Salas-Lais ◽  
Larissa Fernandes-Matano ◽  
Constantino López-Macías ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Neutralizing Antibodies ◽  
Herd Immunity ◽  
Cross Sectional Study ◽  
Molecular Techniques ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cross Sectional ◽  
Neutralizing Activity

Until recently, the incidence of COVID-19 was primarily estimated using molecular diagnostic methods. However, the number of cases is vastly underreported using these methods. Seroprevalence studies estimate cumulative infection incidences and allow monitoring of transmission dynamics, and the presence of neutralizing antibodies in the population. In February 2020, the Mexican Social Security Institute began conducting anonymous unrelated sampling of residual sera from specimens across the country, excluding patients with fever within the previous two weeks and/or patients with an acute respiratory infection. Sampling was carried out weekly and began 17 days before Mexico’s first officially confirmed case. The 24,273 sera obtained were analyzed by chemiluminescent-linked immunosorbent assay (CLIA) IgG S1/S2 and, later, positive cases using this technique were also analyzed to determine the rate of neutralization using the enzyme-linked immunosorbent assay (ELISA). We identified 40 CLIA IgG positive cases before the first official report of SARS-CoV-2 infection in Mexico. The national seroprevalence was 3.5% in February and 33.5% in December. Neutralizing activity among IgG positives patients during overall study period was 86.1%. The extent of the SARS-CoV-2 infection in Mexico is 21 times higher than that reported by molecular techniques. Although the general population is still far from achieving herd immunity, epidemiological indicators should be re-estimated based on serological studies of this type.

scholarly journals High Frequency of Cryptosporidium hominis Infecting Infants Points to A Potential Anthroponotic Transmission in Maputo, Mozambique

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 293
Author(s):  
Idalécia Cossa-Moiane ◽  
Hermínio Cossa ◽  
Adilson Fernando Loforte Bauhofer ◽  
Jorfélia Chilaúle ◽  
Esperança Lourenço Guimarães ◽  
...  
Keyword(s):  
Public Hospitals ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
P Value ◽  
Cryptosporidium Hominis ◽  
Cryptosporidium Spp ◽  
Pcr Rflp ◽  
Maputo City ◽  
Stool Samples ◽  
Polymerase Chain

Cryptosporidium is one of the most important causes of diarrhea in children less than 2 years of age. In this study, we report the frequency, risk factors and species of Cryptosporidium detected by molecular diagnostic methods in children admitted to two public hospitals in Maputo City, Mozambique. We studied 319 patients under the age of five years who were admitted due to diarrhea between April 2015 and February 2016. Single stool samples were examined for the presence of Cryptosporidium spp. oocysts, microscopically by using a Modified Ziehl–Neelsen (mZN) staining method and by using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) technique using 18S ribosomal RNA gene as a target. Overall, 57.7% (184/319) were males, the median age (Interquartile range, IQR) was 11.0 (7–15) months. Cryptosporidium spp. oocysts were detected in 11.0% (35/319) by microscopy and in 35.4% (68/192) using PCR-RFLP. The most affected age group were children older than two years, [adjusted odds ratio (aOR): 5.861; 95% confidence interval (CI): 1.532–22.417; p-value < 0.05]. Children with illiterate caregivers had higher risk of infection (aOR: 1.688; 95% CI: 1.001–2.845; p-value < 0.05). An anthroponotic species C. hominis was found in 93.0% (27/29) of samples. Our findings demonstrated that cryptosporidiosis in children with diarrhea might be caused by anthroponomic transmission.

scholarly journals Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes

2007 ◽  
Vol 88 (2) ◽  
pp. 621-630 ◽  
Author(s):  
S. Maan ◽  
N. S. Maan ◽  
A. R. Samuel ◽  
S. Rao ◽  
H. Attoui ◽  
...  
Keyword(s):  
Bluetongue Virus ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Full Length ◽  
Molecular Diagnostic ◽  
Mammalian Host ◽  
Reading Frame ◽  
Diagnostic Assays ◽  
Primary Determinant ◽  
Serotype Identification

The outer capsid protein VP2 of Bluetongue virus (BTV) is a target for the protective immune response generated by the mammalian host. VP2 contains the majority of epitopes that are recognized by neutralizing antibodies and is therefore also the primary determinant of BTV serotype. Full-length cDNA copies of genome segment 2 (Seg-2, which encodes VP2) from the reference strains of each of the 24 BTV serotypes were synthesized, cloned and sequenced. This represents the first complete set of full-length BTV VP2 genes (from the 24 serotypes) that has been analysed. Each Seg-2 has a single open reading frame, with short inverted repeats adjacent to conserved terminal hexanucleotide sequences. These data demonstrated overall inter-serotype variations in Seg-2 of 29 % (BTV-8 and BTV-18) to 59 % (BTV-16 and BTV-22), while the deduced amino acid sequence of VP2 varied from 22.4 % (BTV-4 and BTV-20) to 73 % (BTV-6 and BTV-22). Ten distinct Seg-2 lineages (nucleotypes) were detected, with greatest sequence similarities between those serotypes that had previously been reported as serologically ‘related’. Fewer similarities were observed between different serotypes in regions of VP2 that have been reported as antigenically important, suggesting that they may play a role in the neutralizing antibody response. The data presented form an initial basis for BTV serotype identification by sequence analyses and comparison of Seg-2, and for development of molecular diagnostic assays for individual BTV serotypes (by RT-PCR).

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