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

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

Multiplex logic processing isothermal diagnostic assays for an evolving virus

10.1101/424440 ◽

2018 ◽

Author(s):

Sanchita Bhadra ◽

Miguel A. Saldaña ◽

Hannah Grace Han ◽

Grant L. Hughes ◽

Andrew D. Ellington

Keyword(s):

Point Of Care ◽

Direct Analysis ◽

Human Saliva ◽

Ease Of Use ◽

Molecular Diagnostic ◽

One Pot ◽

Diagnostic Assays ◽

Specificity And Sensitivity ◽

Isothermal Assay ◽

Fail Safe

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

A field evaluation of a new molecular-based point-of-care test for chlamydia and gonorrhoea in remote Aboriginal health services in Australia

Sexual Health ◽

10.1071/sh14158 ◽

2015 ◽

Vol 12 (1) ◽

pp. 27 ◽

Cited By ~ 14

Author(s):

Louise M. Causer ◽

Belinda Hengel ◽

Lisa Natoli ◽

Annie Tangey ◽

Steven G. Badman ◽

...

Keyword(s):

Health Services ◽

Sensitivity And Specificity ◽

Point Of Care ◽

Simultaneous Detection ◽

Field Evaluation ◽

Aboriginal Health ◽

Ease Of Use ◽

Nucleic Acid Amplification ◽

Specimen Preparation ◽

Sexually Transmissible Infections

Background Point-of-care (POC) tests could be important public health tools in settings with treatment delays and high rates of sexually transmissible infections (STIs). Use is limited due to suboptimal performance. The performance and ease-of-use of a new molecular-based POC test for simultaneous detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) was assessed, alongside two single-organism immunochromatographic tests (ICT). Methods: The evaluation occurred between May 2012 and March 2013 during community STI screens in two remote Aboriginal health services. Urine was tested with the GeneXpert®CT/NG and if sufficient volume, also with Diaquick CT and Gonorrhoea Card. The gold standard comparison was laboratory nucleic acid amplification testing (NAAT). Operational characteristics were also assessed. Results: Among 198 samples, GeneXpert CT sensitivity and specificity was 100% [95% confidence intervals (CI): 75.9–100] and 99.5% (95% CI: 96.5–100), and NG was 100% (95% CI: 96.5–100) and 100% (95% CI: 97.5–100), respectively. Among a sample subset, Diaquick CT (n = 104) sensitivity and specificity was 27.3% (95% CI: 7.3–60.7) and 66.7% (95% CI: 12.5–98.2), and Gonorrhoea Card (n = 29), was 66.7% (95% CI: 12.5–98.2) and 76.9% (95% CI: 56.0–90.2), respectively. GeneXpert required 1 mL of urine, four steps, 1 min specimen preparation and 90 min to result. ICTs required 15 mL of urine, eight steps, 18 min preparation and 10–15 min to result. Conclusion: The accuracy and operational benefits of GeneXpert CT/NG make it very suitable in these settings where delays to treatment are encountered.

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

Multiplexed Prostate Cancer Companion Diagnostic Devices

Sensors ◽

10.3390/s21155023 ◽

2021 ◽

Vol 21 (15) ◽

pp. 5023

Author(s):

Josephine Aidoo-Brown ◽

Despina Moschou ◽

Pedro Estrela

Keyword(s):

Prostate Cancer ◽

Point Of Care ◽

Specific Antigen ◽

Simultaneous Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Protein Biomarkers ◽

Protein Biomarker ◽

Psa Testing ◽

Specificity And Sensitivity ◽

Companion Diagnostic

Prostate cancer (PCa) remains one of the most prominent forms of cancer for men. Since the early 1990s, Prostate-Specific Antigen (PSA) has been a commonly recognized PCa-associated protein biomarker. However, PSA testing has been shown to lack in specificity and sensitivity when needed to diagnose, monitor and/or treat PCa patients successfully. One enhancement could include the simultaneous detection of multiple PCa-associated protein biomarkers alongside PSA, also known as multiplexing. If conventional methods such as the enzyme-linked immunosorbent assay (ELISA) are used, multiplexed detection of such protein biomarkers can result in an increase in the required sample volume, in the complexity of the analytical procedures, and in adding to the cost. Using companion diagnostic devices such as biosensors, which can be portable and cost-effective with multiplexing capacities, may address these limitations. This review explores recent research for multiplexed PCa protein biomarker detection using optical and electrochemical biosensor platforms. Some of the novel and potential serum-based PCa protein biomarkers will be discussed in this review. In addition, this review discusses the importance of converting research protocols into multiplex point-of-care testing (xPOCT) devices to be used in near-patient settings, providing a more personalized approach to PCa patients’ diagnostic, surveillance and treatment management.

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

A Label and Probe-Free Zika Virus Immunosensor Prussian Blue@carbon Nanotube-Based for Amperometric Detection of the NS2B Protein

Biosensors ◽

10.3390/bios11050157 ◽

2021 ◽

Vol 11 (5) ◽

pp. 157

Author(s):

Bárbara V. M. Silva ◽

Marli T. Cordeiro ◽

Marco A. B. Rodrigues ◽

Ernesto T. A. Marques ◽

Rosa F. Dutra

Keyword(s):

Carbon Nanotube ◽

Prussian Blue ◽

Zika Virus ◽

Point Of Care ◽

Amperometric Detection ◽

Cross Reactivity ◽

Structural Protein ◽

Serum Samples ◽

Polypyrrole Composite ◽

Congenital Disabilities

Zika virus (ZIKV) is a mosquito-borne infection, predominant in tropical and subtropical regions causing international concern due to the ZIKV disease having been associated with congenital disabilities, especially microcephaly and other congenital abnormalities in the fetus and newborns. Development of strategies that minimize the devastating impact by monitoring and preventing ZIKV transmission through sexual intercourse, especially in pregnant women, since no vaccine is yet available for the prevention or treatment, is critically important. ZIKV infection is generally asymptomatic and cross-reactivity with dengue virus (DENV) is a global concern. An innovative screen-printed electrode (SPE) was developed for amperometric detection of the non-structural protein (NS2B) of ZIKV by exploring the intrinsic redox catalytic activity of Prussian blue (PB), incorporated into a carbon nanotube–polypyrrole composite. Thus, this immunosensor has the advantage of electrochemical detection without adding any redox-probe solution (probe-less detection), allowing a point-of-care diagnosis. It was responsive to serum samples of only ZIKV positive patients and non-responsive to negative ZIKV patients, even if the sample was DENV positive, indicating a possible differential diagnosis between them by NS2B. All samples used here were confirmed by CDC protocols, and immunosensor responses were also checked in the supernatant of C6/36 and in Vero cell cultures infected with ZIKV.

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

Molecular diagnostic of toxigenic Corynebacterium diphtheriae strain by DNA sensor potentially suitable for electrochemical point-of-care diagnostic

Talanta ◽

10.1016/j.talanta.2021.122161 ◽

2021 ◽

Vol 227 ◽

pp. 122161

Author(s):

Kasper Marchlewicz ◽

Iga Ostrowska ◽

Sławomir Oszwałdowski ◽

Aleksandra Zasada ◽

Robert Ziółkowski ◽

...

Keyword(s):

Point Of Care ◽

Corynebacterium Diphtheriae ◽

Molecular Diagnostic ◽

Dna Sensor

Download Full-text

COVID-19 Biomarkers and Advanced Sensing Technologies for Point-of-Care (POC) Diagnosis

Bioengineering ◽

10.3390/bioengineering8070098 ◽

2021 ◽

Vol 8 (7) ◽

pp. 98

Author(s):

Ernst Emmanuel Etienne ◽

Bharath Babu Nunna ◽

Niladri Talukder ◽

Yudong Wang ◽

Eon Soo Lee

Keyword(s):

Response Times ◽

Rna Virus ◽

Point Of Care ◽

Protein Detection ◽

Turnaround Time ◽

Quantitative Detection ◽

Rt Pcr ◽

Specificity And Sensitivity ◽

Small Market ◽

Multiple Biomarkers

COVID-19, also known as SARS-CoV-2 is a novel, respiratory virus currently plaguing humanity. Genetically, at its core, it is a single-strand positive-sense RNA virus. It is a beta-type Coronavirus and is distinct in its structure and binding mechanism compared to other types of coronaviruses. Testing for the virus remains a challenge due to the small market available for at-home detection. Currently, there are three main types of tests for biomarker detection: viral, antigen and antibody. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) remains the gold standard for viral testing. However, the lack of quantitative detection and turnaround time for results are drawbacks. This manuscript focuses on recent advances in COVID-19 detection that have lower limits of detection and faster response times than RT-PCR testing. The advancements in sensing platforms have amplified the detection levels and provided real-time results for SARS-CoV-2 spike protein detection with limits as low as 1 fg/mL in the Graphene Field Effect Transistor (FET) sensor. Additionally, using multiple biomarkers, detection levels can achieve a specificity and sensitivity level comparable to that of PCR testing. Proper biomarker selection coupled with nano sensing detection platforms are key in the widespread use of Point of Care (POC) diagnosis in COVID-19 detection.

Download Full-text