Multiplex logic processing isothermal diagnostic assays for an evolving virus

AbstractWe have developed a generalizable ‘smart molecular diagnostic’ capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our one-pot isothermal assay relies on multiplex execution of four loop-mediated isothermal amplification reactions, with primers that are degenerate and redundant, thereby increasing the breadth of targets while reducing the probability of amplification failure. An easy-to-read visual answer is computed directly by a multi-input Boolean OR gate signal transducer that uses degenerate strand exchange probes to assess any combination of amplicons. We demonstrate our platform by using the same assay to detect divergent Asian and African lineages of the evolving Zika virus (ZIKV), while maintaining selectivity against non-target viruses. Direct analysis of biological specimens proved possible, with 20 virions / µl being directly detected in human saliva within 90 minutes, and crudely macerated ZIKV-infected Aedes aegypti mosquitoes being identified with 100% specificity and sensitivity. The ease-of-use with minimal instrumentation, broad programmability, and built-in fail-safe reliability make our smart molecular diagnostic attractive for POC use.

Download Full-text

Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay

Viruses ◽

10.3390/v10120714 ◽

2018 ◽

Vol 10 (12) ◽

pp. 714 ◽

Cited By ~ 7

Author(s):

Sanchita Bhadra ◽

Miguel Saldaña ◽

Hannah Han ◽

Grant Hughes ◽

Andrew Ellington

Keyword(s):

Zika Virus ◽

Point Of Care ◽

Logic Gate ◽

Simultaneous Detection ◽

Direct Analysis ◽

Ease Of Use ◽

Molecular Diagnostic ◽

Specificity And Sensitivity ◽

Isothermal Assay ◽

Fail Safe

We have developed a generalizable “smart molecular diagnostic” capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our isothermal assay relies on multiplex execution of four loop-mediated isothermal amplification reactions, with primers that are degenerate and redundant, thereby increasing the breadth of targets while reducing the probability of amplification failure. An easy-to-read visual answer is computed directly by a multi-input Boolean OR logic gate (gate output is true if either one or more gate inputs is true) signal transducer that uses degenerate strand exchange probes to assess any combination of amplicons. We demonstrate our methodology by using the same assay to detect divergent Asian and African lineages of the evolving Zika virus (ZIKV), while maintaining selectivity against non-target viruses. Direct analysis of biological specimens proved possible, with crudely macerated ZIKV-infected Aedes aegypti mosquitoes being identified with 100% specificity and sensitivity. The ease-of-use with minimal instrumentation, broad programmability, and built-in fail-safe reliability make our smart molecular diagnostic attractive for POC use.

Download Full-text

Development of robust isothermal RNA amplification assay for lab-free testing of RNA viruses

Scientific Reports ◽

10.1038/s41598-021-95411-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Radhika Biyani ◽

Kirti Sharma ◽

Kenji Kojima ◽

Madhu Biyani ◽

Vishnu Sharma ◽

...

Keyword(s):

Rna Virus ◽

Point Of Care ◽

Human Saliva ◽

Nucleic Acid Amplification ◽

Potential Game ◽

One Pot ◽

Viral Loads ◽

Copy Numbers ◽

Amplification Assay ◽

Primary Focus

AbstractSimple tests of infectiousness that return results in minutes and directly from samples even with low viral loads could be a potential game-changer in the fight against COVID-19. Here, we describe an improved isothermal nucleic acid amplification assay, termed the RICCA (RNA Isothermal Co-assisted and Coupled Amplification) reaction, that consists of a simple one-pot format of ‘sample-in and result-out’ with a primary focus on the detection of low copy numbers of RNA virus directly from saliva without the need for laboratory processing. We demonstrate our assay by detecting 16S rRNA directly from E. coli cells with a sensitivity as low as 8 CFU/μL and RNA fragments from a synthetic template of SARS-CoV-2 with a sensitivity as low as 1740 copies/μL. We further demonstrate the applicability of our assay for real-time testing at the point of care by designing a closed format for paper-based lateral flow assay and detecting heat-inactivated SARS-COV-2 virus in human saliva at concentrations ranging from 28,000 to 2.8 copies/μL with a total assay time of 15–30 min.

Download Full-text

Competitive SNP-LAMP probes for rapid and robust single-nucleotide polymorphism detection

10.1101/2021.03.29.437576 ◽

2021 ◽

Author(s):

Leland B Hyman ◽

Clare R Christopher ◽

Philip A Romero

Keyword(s):

Point Of Care ◽

High Specificity ◽

Common Source ◽

Lamp Method ◽

Universal Method ◽

Nucleotide Polymorphisms ◽

One Pot ◽

Single Nucleotide ◽

Specificity And Sensitivity ◽

And Shifts

Single-nucleotide polymorphisms (SNPs) are the most common source of genetic variation between individuals and have implications in human disease, pathogen drug resistance, and agriculture. SNPs are typically detected using DNA sequencing, which requires advanced sample preparation and instrumentation, and thus cannot be deployed for on-site testing or in low-resource settings. In this work we have developed a simple and robust assay to rapidly detect SNPs in nucleic acid samples. Our approach combines LAMP-based target amplification with fluorescent probes to detect SNPs with high specificity in a one-pot reaction format. A competitive "sink" strand preferentially binds to off-target products and shifts the free energy landscape to favor specific activation by SNP products. We demonstrated the broad utility and reliability of our SNP-LAMP method by detecting three distinct SNPs across the human genome. We also designed an assay to rapidly detect highly transmissible SARS-CoV-2 variants. This work demonstrates that competitive SNP-LAMP is a powerful and universal method that could be applied in point-of-care settings to detect any target SNP with high specificity and sensitivity.

Download Full-text

Microfluidic Chip with Two-Stage Isothermal Amplification Method for Highly Sensitive Parallel Detection of SARS-CoV-2 and Measles Virus

Micromachines ◽

10.3390/mi12121582 ◽

2021 ◽

Vol 12 (12) ◽

pp. 1582

Author(s):

Qin Huang ◽

Xiaohui Shan ◽

Ranran Cao ◽

Xiangyu Jin ◽

Xue Lin ◽

...

Keyword(s):

Microfluidic Chip ◽

Measles Virus ◽

Point Of Care ◽

Isothermal Amplification ◽

Molecular Diagnostic ◽

Nasopharyngeal Swab ◽

Two Stage ◽

Parallel Detection ◽

Amplification Method ◽

Specificity And Sensitivity

A two-stage isothermal amplification method, which consists of a first-stage basic recombinase polymerase amplification (RPA) and a second-stage fluorescence loop-mediated isothermal amplification (LAMP), as well as a microfluidic-chip-based portable system, were developed in this study; these enabled parallel detection of multiplex targets in real time in around one hour, with high sensitivity and specificity, without cross-contamination. The consumption of the sample and the reagent was 2.1 μL and 10.6 μL per reaction for RPA and LAMP, respectively. The lowest detection limit (LOD) was about 10 copies. The clinical amplification of about 40 nasopharyngeal swab samples, containing 17 SARS-CoV-2 (severe acute respiratory syndrome coronavirus) and 23 measles viruses (MV), were parallel tested by using the microfluidic chip. Both clinical specificity and sensitivity were 100% for MV, and the clinical specificity and sensitivity were 94.12% and 95.83% for SARS-CoV-2, respectively. This two-stage isothermal amplification method based on the microfluidic chip format offers a convenient, clinically parallel molecular diagnostic method, which can identify different nucleic acid samples simultaneously and in a timely manner, and with a low cost of the reaction reagent. It is especially suitable for resource-limited areas and point-of-care testing (POCT).

Download Full-text

Towards effective diagnostic assays for COVID-19: a review

Journal of Clinical Pathology ◽

10.1136/jclinpath-2020-206685 ◽

2020 ◽

Vol 73 (7) ◽

pp. 370-377 ◽

Cited By ~ 22

Author(s):

Marietjie Venter ◽

Karin Richter

Keyword(s):

Point Of Care ◽

Case Definition ◽

Molecular Diagnostic ◽

Public Health Response ◽

Serological Tests ◽

Diagnostic Assays ◽

Laboratory Capacity ◽

Health Response ◽

The Usa ◽

Antigen Tests

Countries globally are affected by the COVID-19 pandemic, with nearly two million cases and 120 000 deaths occurring within 4 months of the discovery of the severe acute respiratory syndrome coronavirus-2 in December 2019 in China. Accurate diagnoses of cases is key in managing the pandemic by identification, isolation and treatment of patients and defining the epidemiology of the virus. By mid-January 2020, a scientist from China published the full genome of the virus, which facilitated the development of accurate molecular diagnostic assays. By the end of January 2020, the WHO, in collaboration with laboratories in Asia, Europe and the USA, published several real-time reverse transcriptase PCR (rtRT-PCR) protocols that allowed identification of cases and development of commercial assays. Clinical investigations facilitated development of accurate case definition and guidance for laboratories on the optimum specimens and procedures for diagnoses. Currently, laboratory-based rtRT-PCR is the recommended test for diagnoses of acute cases to ensure patients can be identified and isolated and to facilitate the public health response. However, due to delays in diagnoses, severe shortage of tests and laboratory capacity, point-of-care molecular or antigen tests are becoming more attractive. Although serological tests are not suitable for diagnoses of acute cases, they are important to define epidemiological questions, including attack rate in the population, and to identify immune individuals. This review aimed to summarise the current available information for diagnoses of cases and to aid laboratories and healthcare workers to select the best assays and procedures.

Download Full-text

High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes

10.1101/2020.04.13.039941 ◽

2020 ◽

Cited By ~ 16

Author(s):

Sanchita Bhadra ◽

Timothy E. Riedel ◽

Simren Lakhotia ◽

Nicholas D. Tran ◽

Andrew D. Ellington

Keyword(s):

Point Of Care ◽

False Negative ◽

Human Saliva ◽

Isothermal Amplification ◽

Nucleic Acid Amplification ◽

Target Sequence ◽

One Pot ◽

Negative Results ◽

False Negative Results ◽

Polymerase Chain

ABSTRACTIsothermal nucleic acid amplification tests (iNAT), such as loop-mediated isothermal amplification (LAMP), are good alternatives to polymerase chain reaction (PCR)-based amplification assays, especially for point-of-care and low resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can generate spurious amplicons, especially in the absence of target sequences, resulting in false positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets, and can accumulate mutations that will lead to inefficient primer binding and thus false negative results. Internally redundant assays targeting different regions of the target sequence can help to reduce such false negatives. Here we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on non-sequence-specific readout into assays that can be visually read using sequence-specific fluorogenic oligonucleotide strand exchange (OSD) probes. We evaluate one-pot operation of both individual and multiplex LAMP-OSD assays and demonstrate detection of SARS-CoV-2 virions in crude human saliva.

Download Full-text

Improving the quality of bacteriological studies for infections caused by carbapenem resistant Klebsiella pneumoniae using molecular diagnostic methods

10.33920/med-06-2001-01 ◽

2020 ◽

pp. 6-17

Author(s):

A. Chizhikova ◽

E. Lisitsyna

Keyword(s):

Klebsiella Pneumoniae ◽

Pathogenic Bacteria ◽

Test System ◽

Ease Of Use ◽

Diagnostic Methods ◽

Molecular Diagnostic ◽

Fast Analysis ◽

Carbapenem Resistant ◽

Specificity And Sensitivity

The resistance of gram-negative pathogenic bacteria to antibiotics of the β-lactam group — carbapenems — is associated with the ability of bacteria to produce carbapenemases. The relevant task is to determine carbapenem-resistant microorganisms to control the spread of resistant strains and conduct effective pharmacotherapy. The results of the development of molecular diagnostic methods, including use of polymerase chain reaction (PCR), for the detection of carbapenem resistant Klebsiella pneumoniae bacteria are presented. A multiplex PCR test system was developed for detecting carbapenem-resistant K. pneumoniae producing KPC and OXA 48-like carbapenemases. The test system is characterized by high specificity and sensitivity, ease of use, fast analysis time (up to 3 hours). Its introduction into clinical and diagnostic practice is promising in terms of improving the quality of bacteriological studies.

Download Full-text

Validation of a point-of-care rapid diagnostic test for hepatitis C for use in resource-limited settings

International Health ◽

10.1093/inthealth/ihy101 ◽

2019 ◽

Vol 11 (4) ◽

pp. 314-315

Author(s):

James S Leathers ◽

Maria Belen Pisano ◽

Viviana Re ◽

Gertine van Oord ◽

Amir Sultan ◽

...

Keyword(s):

Point Of Care ◽

Low Cost ◽

Rapid Diagnosis ◽

Direct Acting Antivirals ◽

Resource Limited Settings ◽

Rapid Diagnosis Test ◽

Resource Limited ◽

Specificity And Sensitivity ◽

Hcv Screening ◽

Direct Acting

Abstract Background Treatment of HCV with direct-acting antivirals has enabled the discussion of HCV eradication worldwide. Envisioning this aim requires implementation of mass screening in resource-limited areas, usually constrained by testing costs. Methods We validated a low-cost, rapid diagnosis test (RDT) for HCV in three different continents in 141 individuals. Results The HCV RDT showed 100% specificity and sensitivity across different samples regardless of genotype or viral load (in samples with such information, 90%). Conclusions The HCV test validated in this study can allow for HCV screening in areas of need when properly used.

Download Full-text

Comparison of Four Commercially Available Point-of-Care Tests to Detect Antibodies against Canine Parvovirus in Dogs

Viruses ◽

10.3390/v13010018 ◽

2020 ◽

Vol 13 (1) ◽

pp. 18

Author(s):

Michèle Bergmann ◽

Mike Holzheu ◽

Yury Zablotski ◽

Stephanie Speck ◽

Uwe Truyen ◽

...

Keyword(s):

Test Performance ◽

Point Of Care ◽

Reference Method ◽

Canine Parvovirus ◽

P Value ◽

Important Indicator ◽

Specificity And Sensitivity ◽

Specific Pathogen ◽

The One ◽

Point Of Care Tests

Measuring antibodies to evaluate dogs´ immunity against canine parvovirus (CPV) is useful to avoid unnecessary re-vaccinations. The study aimed to evaluate the quality and practicability of four point-of-care (POC) tests for detection of anti-CPV antibodies. The sera of 198 client-owned and 43 specific pathogen-free (SPF) dogs were included; virus neutralization was the reference method. Specificity, sensitivity, positive and negative predictive value (PPV and NPV), and overall accuracy (OA) were calculated. Specificity was considered to be the most important indicator for POC test performance. Differences between specificity and sensitivity of POC tests in the sera of all dogs were determined by McNemar, agreement by Cohen´s kappa. Prevalence of anti-CPV antibodies in all dogs was 80% (192/241); in the subgroup of client-owned dogs, it was 97% (192/198); and in the subgroup of SPF dogs, it was 0% (0/43). FASTest® and CanTiCheck® were easiest to perform. Specificity was highest in the CanTiCheck® (overall dogs, 98%; client-owned dogs, 83%; SPF dogs, 100%) and the TiterCHEK® (overall dogs, 96%; client-owned dogs, 67%; SPF dogs, 100%); no significant differences in specificity were observed between the ImmunoComb®, the TiterCHEK®, and the CanTiCheck®. Sensitivity was highest in the FASTest® (overall dogs, 95%; client-owned dogs, 95%) and the CanTiCheck® (overall dogs, 80%; client-owned dogs, 80%); sensitivity of the FASTest® was significantly higher compared to the one of the other three tests (McNemars p-value in each comparison: <0.001). CanTiCheck® would be the POC test of choice when considering specificity and practicability. However, differences in the number of false positive results between CanTiCheck®, TiterCHEK®, and ImmunoComb® were minimal.

Download Full-text