scholarly journals Rapid Ultrasensitive Detection of Clostridiodes difficile Toxins in Stool Samples Using A Single-Molecule Counting Method

2020 ◽  
Vol 41 (S1) ◽  
pp. s450-s451
Author(s):  
Don Straus ◽  
Ann Zuniga ◽  
Alejandra Garces ◽  
Andrew Tempesta ◽  
Adam Williams ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Reference Method ◽  
The United States ◽  
Nucleic Acid Amplification ◽  
Specimen Preparation ◽  
Analytical Sensitivity ◽  
Percentage Agreement ◽  
Toxin B ◽  
Spore Form ◽  
Stool Samples

Background:Clostridiodes difficile infection is considered an urgent antibiotic resistance threat by the CDC, accounting for ∼225,000 hospitalizations, 12,800 deaths, and ∼$1 billion in healthcare costs in the United States in 2017. The presence of the secreted toxins that cause the devastating symptoms of this gastrointestinal infection are diagnostic of C. difficile infection (CDI). However, the rapid testing methods currently used to detect CDI lack accuracy. Enzyme immunoassays are specific but lack sensitivity because they do not detect CDI patients that have low levels of the toxins. Nucleic acid amplification tests (NAATs) are sensitive, but they lack specificity because they detect patients colonized with C. difficile in the dormant spore form that does not produce the toxins. This insufficiency has resulted in the adoption of complex multitest algorithms for C. difficile diagnosis. We present results for a new toxin test that demonstrates both high clinical sensitivity and clinical specificity for C. difficile toxin B on a fully automated benchtop platform. Methods: The detection technology uses nonmagnified digital imaging to count single toxin molecules that tether together target-specific magnetic and fluorescent particles. The 30-minute method includes the use of a dye cushion to eliminate wash steps and the need for time-consuming specimen preparation steps. We determined analytical performance characteristics of the test using negative clinical stool samples spiked with purified toxin. To assess clinical performance, we tested 785 stool samples from 5 clinical sites and compared the results with the cellular cytotoxicity neutralization assay (CCNA). Results: The test’s limit of detection for toxin B was 60 pg/mL. A comparison of the new test to the CCNA reference method gave 98% positive percentage agreement (83 of 85 samples) and 95% negative percentage agreement (667 of 700 samples). Conclusions: The new method demonstrated 96% accuracy compared to the gold standard for C. difficile toxin tests. The results also demonstrate an analytical sensitivity (limit of detection, 60 pg/mL). Thus, the test has the potential to detect CDI patients missed by enzyme immunoassay (EIA) tests due to their low analytical sensitivity. Because the test detects toxins directly, it is expected to have a lower false-positive rate than NAAT methods, which detect patients colonized with the non–toxin-producing spore form. A single accurate test for toxin-producing C. difficile could eliminate the need for multitest algorithms.Funding: First Light Diagnostics, Inc., provided support for this study.Disclosures: Donald Straus reports that he is the founder and chief scientific officer of First Light Diagnostics (FLDx) with salary and ownership interest in the form of stocks, stock options, and warrants. Adam Williams reports salary from First Light Diagnostics.

scholarly journals Development and Validation of Digital Enzyme-Linked Immunosorbent Assays for Ultrasensitive Detection and Quantification of Clostridium difficile Toxins in Stool

2015 ◽  
Vol 53 (10) ◽  
pp. 3204-3212 ◽  
Author(s):  
Linan Song ◽  
Mingwei Zhao ◽  
David C. Duffy ◽  
Joshua Hansen ◽  
Kelsey Shields ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Cytotoxicity Assay ◽  
Nucleic Acid Amplification ◽  
Analytical Sensitivity ◽  
Toxin B ◽  
Content Type ◽  
Toxigenic Culture ◽  
Immunosorbent Assays ◽  
Digital Elisa ◽  
Detection And Quantification

The currently available diagnostics forClostridium difficileinfection (CDI) have major limitations. Despite mounting evidence that toxin detection is paramount for diagnosis, conventional toxin immunoassays are insufficiently sensitive and cytotoxicity assays too complex; assays that detect toxigenic organisms (toxigenic culture [TC] and nucleic acid amplification testing [NAAT]) are confounded by asymptomatic colonization by toxigenicC. difficile. We developed ultrasensitive digital enzyme-linked immunosorbent assays (ELISAs) for toxins A and B using single-molecule array technology and validated the assays using (i) culture filtrates from a panel of clinicalC. difficileisolates and (ii) 149 adult stool specimens already tested routinely by NAAT. The digital ELISAs detected toxins A and B in stool with limits of detection of 0.45 and 1.5 pg/ml, respectively, quantified toxins across a 4-log range, and detected toxins from all clinical strains studied. Using specimens that were negative by cytotoxicity assay/TC/NAAT, clinical cutoffs were set at 29.4 pg/ml (toxin A) and 23.3 pg/ml (toxin B); the resulting clinical specificities were 96% and 98%, respectively. The toxin B digital ELISA was 100% sensitive versus cytotoxicity assay. Twenty-five percent and 22% of the samples positive by NAAT and TC, respectively, were negative by the toxin B digital ELISA, consistent with the presence of organism but minimal or no toxin. The mean toxin levels by digital ELISA were 1.5- to 1.7-fold higher in five patients with CDI-attributable severe outcomes, versus 68 patients without, but this difference was not statistically significant. Ultrasensitive digital ELISAs for the detection and quantification of toxins A and B in stool can provide a rapid and simple tool for the diagnosis of CDI with both high analytical sensitivity and high clinical specificity.

scholarly journals 2359. Prospective Feasibility Study for Novel Ultrasensitive Multiplexed Immunoassay for Clostridiodes difficile Toxins A and B

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S812-S813
Author(s):  
Lauren Watson ◽  
Michele L Zimbric ◽  
Catherine Shaughnessy ◽  
Shraddha Kale ◽  
Jeff Debad ◽  
...  
Keyword(s):  
Diagnostic Tests ◽  
Standard Of Care ◽  
Positive Sample ◽  
Nucleic Acid Amplification ◽  
Serum Samples ◽  
Toxin A ◽  
Toxin B ◽  
Diagnostic Assays ◽  
Stool Samples ◽  
Multiplexed Immunoassay

Abstract Background The diagnosis of Clostridiodes difficile infection is challenging. A wide array of diagnostic tests are used in practice; however, each available test has important limitations. We examined the feasibility and analytical performance of a novel ultrasensitive multiplexed immunoassay designed by Meso Scale Diagnostics (MSD) compared with five current diagnostic assays for detection of C. difficile toxin A and B. Methods Stool, serum and urine samples from 44 admitted inpatients were collected within 72 hours of a standard of care nucleic acid amplification test (NAAT) result (23 positive, 21 negative). These specimens underwent five standard diagnostic assays: enzyme immunoassay for toxins A and B (EIA), cytotoxin cell assay, bacterial culture isolation, and two different NAATs to determine presence of viable C. difficile cells, toxins, and toxin-encoding genes (Table 1). The concentration (fg/mL) of toxin A and toxin B in all stool samples was then quantified using MSD’s multiplexed immunoassay (Table 1). Results At least one of the five standard diagnostic tests for C. difficile was positive in 16 of the 23 clinically positive patients. The MSD multiplex immunoassay detected toxin A and/or toxin B in 15 of these 16 samples and quantified low levels of toxin A in one clinically positive sample that was negative for all other tests. In contrast, only 2 of the 16 positive samples were positive by EIA, demonstrating the benefits of the ultrasensitive assay over standard immunoassay methods. All clinically negative specimens were negative in all tests. Toxin detection in urine and serum samples was negligible. In stool samples, the MSD test had an estimated sensitivity of 93% (95% CI: 70–99%) and specificity of 93% (95% CI: 78–98%) compared with the clinically used NAAT. Conclusion The MSD multiplex toxin assay is a feasible test to move forward for further evaluation. Ultimately, future studies should examine the performance of this test compared with standard of care in a prospective randomized trial assessing clinical outcomes. Disclosures All authors: No reported disclosures.

Assessment of Commercial Real-Time PCR Assays for Detection of Malaria Infection in a Non-Endemic Setting

2021 ◽  
Author(s):  
Alexandra Martín Ramírez ◽  
Thuy Huong Ta Tang ◽  
Marta Lanza Suárez ◽  
Ana Álvarez Fernández ◽  
Carlota Muñoz García ◽  
...  
Keyword(s):  
Real Time ◽  
Malaria Control ◽  
Limit Of Detection ◽  
Reference Method ◽  
Malaria Diagnosis ◽  
Mean Value ◽  
Analytical Sensitivity ◽  
Accurate Treatment ◽  
Pcr Assays ◽  
Good Concordance

Malaria control and elimination require prompt diagnosis and accurate treatment. Conventional methods such as rapid diagnostic tests (RDTs) and microscopy lack the characteristics to detect low parasitemias, commonly found in asymptomatic parasitemias and/or submicroscopic malaria carriers. On the contrary, molecular methods have higher sensitivity and specificity. This study evaluated the performance of two commercial real-time polymerase chain reaction (PCR) assays, RealStar® Malaria PCR (RealStar-genus) and RealStar Malaria Screen&Type PCR (RealStar-species), compared with the reference Nested Multiplex Malaria PCR, for the detection of the main five Plasmodium species affecting humans. A total of 121 samples were evaluated. Values of sensitivity (98.9% and 97.8%) and specificity (100% and 96.7%) of the RealStar-genus and the RealStar-species assays, respectively, were very good. The limit of detection (LoD) for the RealStar-genus assay showed a mean value of 0.28 parasites/µL with Plasmodium falciparum samples; while, the LoD of the RealStar-species assay ranged from 0.09 parasites/µL for P. vivax to two parasites/µL for P. ovale. The time to complete a diagnosis was established in 4 hours. Our findings showed a very good concordance of both assays compared with the reference method, with a very good analytical sensitivity. RealStar-species assay was able to correctly characterize double and triple infections. Therefore, these RealStar assays have shown to be useful tools in malaria diagnosis in non-endemic countries and even endemic countries, and for malaria control in general, detecting low parasitemias with sensitivity similar to the most sensitive methods as nested PCR, but with lower time to get the results.

Detection of Non-O157 Shiga Toxin–Producing Escherichia coli in 375 Grams of Beef Trim Enrichments across Multiple Commercial PCR Detection Platforms

2015 ◽  
Vol 78 (1) ◽  
pp. 196-202 ◽  
Author(s):  
SARITA RAENGPRADUB WHEELER ◽  
PRECIAUS HEARD ◽  
CHRISTOPHE DUFOUR ◽  
DELPHINE THEVENOT-SERGENTET ◽  
ESTELLE LOUKIADIS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Limit Of Detection ◽  
Reference Method ◽  
Immunomagnetic Separation ◽  
The United States ◽  
Detection Methods ◽  
Primary Concern ◽  
The European Union ◽  
Commercial Method

Although serotype O157:H7 remains the pathogenic Shiga toxin–producing Escherichia coli (STEC) of primary concern worldwide, some focus in the United States has shifted to six particular non-O157 STEC serogroups (O26, O45, O103, O111, O121, and O145). Some of these serogroups have also emerged as concerns elsewhere around the world, including Europe. The objective of this work was to compare commercial detection methods with the U.S. Department of Agriculture (USDA) reference method for detection of non-O157 STEC in 375 g of beef trim using a limit of detection study design. Overall, the commercial platforms performed well, showing similar levels of sensitivity for detection of presumptive positives for O45, O26, O103, and O121 (PCR screen results only). For O111, one method that utilizes an integrated immunomagnetic separation and PCR approach was more sensitive than a PCR-only screen approach. Additionally, one commercial method showed more presumptive and confirmed positives overall. Use of an immunomagnetic separation tool, such as antibody-coated beads, aided considerably with the confirmation procedures and is an important step when confirming suspect samples. A secondary goal of this study was to evaluate isolation and International Organization for Standardization confirmation protocols used in Europe compared with strategies provided by the USDA Microbiology Laboratory Guidebook (MLG). Generally, results from the USDA confirmation plates (modified Rainbow agar) were better than the European Union confirmation plates (MacConkey agar with or without rhamnose). In summary, detection of non-O157 STEC in 375 g of beef trim can be performed by any of the three methods on the market evaluated in the study.

scholarly journals Performing Reflexive Toxin A/B Enzyme Immunoassay in a Two-Step Algorithm for the Diagnosis of Clostridium difficile Infection Has Limited Clinical Utility

2018 ◽  
Author(s):  
M Mounajjed ◽  
R Pease ◽  
B Jung-Hynes ◽  
N Safdar ◽  
D Chen
Keyword(s):  
Clostridium Difficile ◽  
Enzyme Immunoassay ◽  
Electronic Medical Records ◽  
Medical Records ◽  
Clinical Utility ◽  
Nucleic Acid Amplification ◽  
Toxin B ◽  
Clinical Sensitivity ◽  
Stool Samples ◽  
Step Algorithm

ABSTRACTThe differentiation of Clostridium difficile infection (CDI) from colonization is challenged by the suboptimal clinical specificity of nucleic acid amplification tests (NAAT). In this study, we examined the utility of testing for toxin via enzyme immunoassay (EIA) in specimens already tested by NAAT for the diagnosis of CDI in an attempt to differentiate colonization from infection. We tested 59 stool samples for the presence of C. difficile toxin B gene by NAAT followed by EIAs for glutamate dehydrogenase (GDH EIA) and toxins A and B (Toxin EIA). Two infectious disease physicians independently reviewed the patients’ electronic medical records retrospectively to categorize each patient as CDI-Likely, CDI-Unlikely, or CDI-Indeterminate. Clinical sensitivities and specificities were calculated using 3 definitions of “true” CDI status, being: (1) concordance between both reviewers, (2) concordance, and CDI-Indeterminate/discordant cases classified as CDI-Likely, and (3) concordance, and CDI-Indeterminate/discordant cases classified as CDI-Unlikely. Based on these definitions, clinical sensitivity and specificity for NAAT was 100% and 49-94%, GDH EIA was 83-85% and 43-89%, and Toxin EIA was 39-42% and 83-100%, respectively. 85% (22 of 26) of patients who were NAAT-positive but Toxin EIA-negative symptomatically benefited from treatment for CDI. The addition of EIA to NAAT for CDI diagnosis had limited utility for differentiating colonization from CDI and could have led to under treatment of patients with CDI.

scholarly journals Ultrasensitive Detection of Clostridioides difficile Toxins A and B by Use of Automated Single-Molecule Counting Technology

2018 ◽  
Vol 56 (11) ◽  
Author(s):  
Johanna Sandlund ◽  
Amelita Bartolome ◽  
Anna Almazan ◽  
Stanley Tam ◽  
Sheryl Biscocho ◽  
...  
Keyword(s):  
Single Molecule ◽  
Neutralization Assay ◽  
Cross Reactivity ◽  
Nucleic Acid Amplification ◽  
Analytical Sensitivity ◽  
Content Type ◽  
Testing Procedures ◽  
Clostridioides Difficile ◽  
A Cell ◽  
Stool Samples

ABSTRACT Current tests for the detection of Clostridioides (formerly Clostridium) difficile free toxins in feces lack sensitivity, while nucleic acid amplification tests lack clinical specificity. We have evaluated the Singulex Clarity C. diff toxins A/B assay (currently in development), an automated and rapid ultrasensitive immunoassay powered by single-molecule counting technology, for detection of C. difficile toxin A (TcdA) and toxin B (TcdB) in stool. The analytical sensitivity, analytical specificity, repeatability, and stability of the assay were determined. In a clinical evaluation, frozen stool samples from 311 patients with suspected C. difficile infection were tested with the Clarity C. diff toxins A/B assay, using an established cutoff value. Samples were tested with the Xpert C. difficile/Epi assay, and PCR-positive samples were tested with an enzyme immunoassay (EIA) (C. Diff Quik Chek Complete). EIA-negative samples were further tested with a cell cytotoxicity neutralization assay. The limits of detection for TcdA and TcdB were 0.8 and 0.3 pg/ml in buffer and 2.0 and 0.7 pg/ml in stool, respectively. The assay demonstrated reactivity to common C. difficile strains, did not show cross-reactivity to common gastrointestinal pathogens, was robust against common interferents, allowed detection in fresh and frozen stool samples and in samples after three freeze-thaw cycles, and provided results with high reproducibility. Compared to multistep PCR and toxin-testing procedures, the Singulex Clarity C. diff toxins A/B assay yielded 97.7% sensitivity and 100% specificity. The Singulex Clarity C. diff toxins A/B assay is ultrasensitive and highly specific and may offer a standalone solution for rapid detection and quantitation of free toxins in stool.

scholarly journals Evaluation of the cobas Cdiff Test for Detection of Toxigenic Clostridium difficile in Stool Samples

2017 ◽  
Vol 55 (12) ◽  
pp. 3426-3436 ◽  
Author(s):  
Lance R. Peterson ◽  
Stephen A. Young ◽  
Thomas E. Davis ◽  
Zi-Xuam Wang ◽  
John Duncan ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
False Negative ◽  
Reference Method ◽  
Culture Method ◽  
Nucleic Acid Amplification ◽  
Culture Methods ◽  
Content Type ◽  
Toxigenic Culture ◽  
Discrepancy Analysis ◽  
Stool Samples

ABSTRACTNucleic acid amplification tests (NAATs) are reliable tools for the detection of toxigenicClostridium difficilefrom unformed (liquid or soft) stool samples. The objective of this study was to evaluate performance of the cobas Cdiff test on the cobas 4800 system using prospectively collected stool specimens from patients suspected of havingC. difficileinfection (CDI). The performance of the cobas Cdiff test was compared to the results of combined direct and broth-enriched toxigenic culture methods in a large, multicenter clinical trial. Additional discrepancy analysis was performed by using the XpertC. difficileEpi test. Sample storage was evaluated by using contrived and fresh samples before and after storage at −20°C. Testing was performed on samples from 683 subjects (306 males and 377 females); 113 (16.5%) of 683 subjects were positive for toxigenicC. difficileby direct toxigenic culture, and 141 of 682 subjects were positive by using the combined direct and enriched toxigenic culture method (reference method), for a prevalence rate of 20.7%. The sensitivity and specificity of the cobas Cdiff test compared to the combined direct and enriched culture method were 92.9% (131/141; 95% confidence interval [CI], 87.4% to 96.1%) and 98.7% (534/541; 95% CI, 97.4% to 99.4%), respectively. Discrepancy analysis using results for retested samples from a second NAAT (Xpert C. difficile/Epi test; Cepheid, Sunnyvale, CA) found no false-negative and 4 false-positive cobas Cdiff test results. There was no difference in positive and negative results in comparisons of fresh and stored samples. These results support the use of the cobas Cdiff test as a robust aid in the diagnosis of CDI.

scholarly journals 730. Severity Of Clostridioides difficile Infection Based On Toxin Analysis, Acid Suppressant Medications and Antibiotics

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S415-S416
Author(s):  
Gayathri Krishnan ◽  
Richa Parikh ◽  
Anna N Witt ◽  
Kulsum Bano ◽  
Sudeepa Bhattacharyya ◽  
...  
Keyword(s):  
Health Care ◽  
Medical Center ◽  
The United States ◽  
Nucleic Acid Amplification ◽  
Clinical Severity ◽  
Toxin Gene ◽  
Antibiotic Exposure ◽  
Toxin B ◽  
Major Health Problem ◽  
Clostridioides Difficile

Abstract Background Clostridioides difficile (C difficile) infection (CDI) is a major health problem in the United States and despite updated guidelines, the laboratory diagnosis remains vexed. A multistep algorithm is recommended to diagnose CDI that includes antigen, toxin and toxin gene Nucleic Acid Amplification (NAAT) assays. This study was done to assess severity of CDI based on toxin B and NAAT statuses. The other objective was to analyze if antibiotics and PPI/H2B (Proton Pump Inhibitors and H2 blockers) affected severity of CDI. Methods Retrospective analysis of all adult patients admitted to a tertiary medical center with diarrhea and a positive C difficile antigen test from 01/2017- 12/2017. From more than 2000 stool samples submitted to the lab, C diff antigen was positive in 265 patients. 191 were diagnosed with CDI based on the 2-step algorithm. Clinical data was available for 168 patients. Severity of CDI was determined based on published guidelines. Fischer’s exact test was used for statistical analysis. Results The mean age at diagnosis was 55.96. Toxin B was detected in 34% (57/168) patients and Toxin NAAT positive in 66% (111/168) patients. 57% of CDI was health care onset compared to 43% with community onset. 42% (72/168) were classified as severe out of which 40.2% (29) were toxin B positive, and 59.8% (43) were NAAT positive. There were no significant differences in severity of CDI based on toxin B and NAAT status (50.9% vs 38.4%, p=0.14). 46% of cases from community vs 39.6% from hospitals were classified as severe CDI (p=0.415). 72% of cases had antibiotic use in the last 30 days. Use of antibiotics was significantly associated with severe CDI (82% vs 64%, p=0.015). 62.5% (105) patients had history of PPI/H2B use and severity was not significantly associated with its use (p=0.872). Conclusion Our study shows that the presence of toxin did not significantly impact the clinical severity of CDI. The use of antibiotics did not affect the presence of toxin although the total number of CDI cases with previous antibiotic exposure was high. Patients who had recent antibiotic exposure were more likely to have severe clinical presentation. More toxin positive cases were health care onset but the effect was not pronounced. Severity of CDI did not significantly depend on health care onset or on exposure to PPI/H2B. Disclosures Atul Kothari, MD, Ansun Biopharma (Consultant)

scholarly journals Comparative performance of four nucleic acid amplification tests for SARS-CoV-2 virus

2020 ◽  
Author(s):  
Yujuan Xiong ◽  
Zhen-Zhen Li ◽  
Qi-Zhen Zhuang ◽  
Yan Chao ◽  
Fei Li ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
High Specificity ◽  
Nucleic Acid Amplification ◽  
Virus Concentration ◽  
Analytical Sensitivity ◽  
Comparative Performance ◽  
Consistency Test ◽  
Nucleic Acid Amplification Tests ◽  
Selection Of

AbstractCoronavirus disease 2019 (COVID-19) can be screened and diagnosed through the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by real-time reverse transcription polymerase chain reaction. SARS-CoV-2 nucleic acid amplification tests (NAATs) have been rapidly developed and quickly applied to clinical testing during the pandemic. However, studies evaluating the performance of these NAAT assays are limited. We evaluated the performance of four NAATs, which were marked by the Conformité Européenne and widely used in China during the pandemic. Results showed that the analytical sensitivity of the four assays was significantly lower than that claimed by the NAAT manufacturers. The limit of detection (LOD) of Daan, Sansure, and Hybribio NAATs was 3000 copies/mL, whereas the LOD of Bioperfectus NAATs was 4000 copies/mL. The results of the consistency test using 46 samples showed that Daan, Sansure, and Hybribio NAATs could detect the samples with a specificity of 100% (30/30) and a sensitivity of 100% (16 /16), whereas Bioperfectus NAAT detected the samples with a specificity of 100% (30/30) and a sensitivity 81.25% (13/16). The sensitivity of Bioperfectus NAAT was lower than that of the three other NAATs; this finding was consistent with the result that Bioperfectus NAAT had a higher LOD than the three other kinds of NAATs. The four above mentioned reagents presented high specificity; however, for the detection of the samples with low virus concentration, Bioperfectus reagent had the risk of missing detection. Therefore, the LOD should be considered in the selection of SARS-CoV-2 NAATs.

scholarly journals Rapid Detection of Hepatitis B Virus in Blood Samples Using a Combination of Polymerase Spiral Reaction With Nanoparticles Lateral-Flow Biosensor

2021 ◽  
Vol 7 ◽  
Author(s):  
Lin Lin ◽  
Jinshuai Guo ◽  
Haiyang Liu ◽  
Xiaofeng Jiang
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Diagnostic Technique ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Nucleic Acid Amplification ◽  
Analytical Sensitivity ◽  
Blood Samples ◽  
Nucleic Acid Amplification Technology

A rapid, highly sensitive, and robust diagnostic technique for point-of-care (PoC) testing can be developed using the combination of the nanoparticle-based lateral flow biosensors (LFB) and isothermal nucleic acid amplification technology. Here, we developed a polymerase spiral reaction (PSR) containing FITC-labeled DNA probes coupled with the nanoparticle-based LFB assay (PSR-LFB) to detect the amplified products to detect HBV visually. Under the optimized conditions, the PSR assay involved incubation of the reaction mixture for 20 min at 63°C, followed by visual detection of positive amplicons using LFB, which would generate a red test line based on the biotin/streptavidin interaction and immunoreactions, within 5 min. A cross-reactivity test revealed that the developed PSR-LFB assay showed good specificity for HBV and could distinguish HBV from other pathogenic microorganisms. For the analytical sensitivity, the limit of detection (LoD) of PSR-LFB assay was recorded as 5.4 copies/mL of HBV genomic DNA, which was ten-times more sensitive than qPCR and loop-mediated isothermal amplification (LAMP). Additionally, all the HBV-positive (29/82) samples, identified using ELISA, were also successfully detected by the PSR-LFB assay. We found that the true positive rate of the PSR-LFB assay was higher than that of qPCR (100 vs. 89.66%, respectively), as well as the LAMP assay (100 vs. 96.55%, respectively). Furthermore, the integrated procedure could be completed in 60 min, including the processing of the blood samples (30 min), an isothermal reaction (20 min), and result visualization (5 min). Thus, this PSR-LFB assay could be a potentially useful technique for PoC diagnosis of HBV in resource-limited countries.

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