scholarly journals Contamination-resistant, rapid emulsion-based isothermal nucleic acid amplification with Mie-scatter inspired light scatter analysis for bacterial identification

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexander S. Day ◽  
Tiffany-Heather Ulep ◽  
Elizabeth Budiman ◽  
Laurel Dieckhaus ◽  
Babak Safavinia ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Amplification ◽  
Nucleic Acid Detection ◽  
Light Scatter ◽  
Emulsion Droplets ◽  
Assay Performance ◽  
Bacterial Adsorption ◽  
Scatter Intensity ◽  
The Impact ◽  
Theory Light

AbstractAn emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to reduce the impact that contamination has on assay performance. Ongoing LAMP reactions within the emulsion droplets cause a decrease in interfacial tension, causing a decrease in droplet size, which results in decreased light scatter intensity due to Mie theory. Light scatter intensity was monitored via spectrophotometers and fiber optic cables placed at 30° and 60°. Light scatter intensities collected at 3 min, 30° were able to statistically differentiate 103 and 106 CFU/µL initial Escherichia coli O157:H7 concentrations compared to NTC (0 CFU/µL), while the intensity at 60° were able to statistically differentiate 106 CFU/µL initial concentrations and NTC. Control experiments were conducted to validate nucleic acid detection versus bacterial adsorption, finding that the light scatter intensities change is due specifically to ongoing LAMP amplification. After inducing contamination of bulk LAMP reagents, specificity lowered to 0% with conventional LAMP, while the eLAMP platform showed 87.5% specificity. We have demonstrated the use of angle-dependent light scatter intensity as a means of real-time monitoring of an emulsion LAMP platform and fabricated a smartphone-based monitoring system that showed similar trends as spectrophotometer light scatter data, validating the technology for a field deployable platform.

scholarly journals Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method

2020 ◽  
Author(s):  
Weihua Yang ◽  
Xiaofei Dang ◽  
Qingxi Wang ◽  
Mingjie Xu ◽  
Qianqian Zhao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Virus Disease ◽  
Lamp Assay ◽  
Nucleic Acid Amplification ◽  
N Gene ◽  
Nucleic Acid Detection ◽  
Lamp Method ◽  
Rt Pcr ◽  
Rapid Amplification

AbstractCorona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. Real-time fluorescent PCR (RT-PCR) is the clinical standard for SARS-CoV-2 nucleic acid detection. To detect SARS-CoV-2 early and control the disease spreading on time, a faster and more convenient method for SARS-CoV-2 nucleic acid detecting, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. RNA reverse transcription and nucleic acid amplification were performed in one step at 63 °C isothermal conditions, and the results can be obtained within 30 minutes. ORF1ab gene, E gene and N gene were detected at the same time. ORF1ab gene was very specific and N gene was very sensitivity, so they can guarantee both sensitivity and specificity for SARS-CoV-2. The sensitivity of RT-LAMP assay is similar to RT-PCR, and specificity was 99% as detecting 208 clinical specimens. The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.

Transition from Antigenemia to Quantitative Nucleic Acid Amplification Testing in Kidney Transplant Recipients Receiving Preemptive Therapy for Cytomegalovirus Infection.

2021 ◽  
Author(s):  
Mônica Rika Nakamura ◽  
Lúcio R. Requião-Moura ◽  
Roberto Mayer Gallo ◽  
Camila Botelho ◽  
Júlia Taddeo ◽  
...  
Keyword(s):  
Glomerular Filtration Rate ◽  
Nucleic Acid ◽  
Acute Rejection ◽  
Glomerular Filtration ◽  
Filtration Rate ◽  
Nucleic Acid Amplification ◽  
Rt Pcr ◽  
Cmv Infection ◽  
Preemptive Treatment ◽  
The Impact

Abstract Due to the high costs, the strategy to reduce the impact of cytomegalovirus (CMV) after kidney transplant (KT) involves preemptive treatment in low and middle-income countries. Thus, this retrospective cohort study compared the performance of antigenemia transitioned to quantitative nucleic acid amplification testing, RT-PCR, in KT recipients receiving preemptive treatment as a strategy to prevent CMV infection. Between 2016 and 2018, 363 patients were enrolled and received preemptive treatment based on antigenemia (n=177) or RT-PCR (n=186). The primary outcome was CMV infection or disease. There were no differences in one-year cumulative incidence of CMV-related events (50.8% vs. 44.1%, P=0.20), neither in time to diagnosis (47.0 vs. 47.0 days) among patients conducted by antigenemia vs. RT-PCR, respectively. The length of CMV first treatment was longer with RT-PCR (20.0 vs. 27.5 days, P<0.001), while the rate of retreatment was not different (14.7% vs. 11.8%, P=0.48). In the Cox regression, the variables associated with CMV-related events were acute rejection within 30 days (HR=2.05, p=0.01) and 30-day glomerular filtration rate (HR=0.98, p<0.001). In conclusion, acute rejection and glomerular filtration rate are risk factors for CMV infection and disease, showing comparable performance in the impact of CMV-related events between antigenemia and RT-PCR for preemptive treatment.

scholarly journals IsoPCR: An Analytically Sensitive, Nested, Multiplex Nucleic Acid Amplification Method

2013 ◽  
Vol 59 (2) ◽  
pp. 436-439 ◽  
Author(s):  
Martin Jensen Søe ◽  
Mikkel Rohde ◽  
Jens Mikkelsen ◽  
Peter Warthoe
Keyword(s):  
Nucleic Acid ◽  
Candida Glabrata ◽  
Simultaneous Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Qpcr Assay ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Nucleic Acid Detection ◽  
Amplification Method

BACKGROUND Nucleic acid tests that can simultaneously detect multiple targets with high sensitivity, specificity, and speed are highly desirable. To meet this need, we developed a new approach we call the isoPCR method. METHODS The isoPCR method is a 2-stage nested-like nucleic acid amplification method that combines a single multiplex preamplification PCR with subsequent distinct detection of specific targets by use of isothermal amplification. We compared isoPCR to nested quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP), and nested LAMP (PCR followed by LAMP), for detection of DNA from Candida glabrata. We evaluated the method's multiplex capability for detecting low copy numbers of pathogens commonly involved in sepsis. RESULTS IsoPCR provided detection of 1 copy of Candida glabrata, an LOD that was 5-fold lower than a nested qPCR assay (5 copies), while the amplification time was simultaneously halved. Similarly, the LOD for isoPCR was lower than that for a LAMP assay (1000 copies) and a nested LAMP assay (5 copies). IsoPCR required recognition of 6 regions for detection, thereby providing a theoretically higher specificity compared to nested qPCR (4 regions). The isoPCR multiplexing capability was demonstrated by simultaneous detection of 4 pathogens with individual LODs of 10 copies or fewer. Furthermore, the specificity of isoPCR was demonstrated by successful pathogen detection from samples with more than 1 pathogen present. CONCLUSIONS IsoPCR provides a molecular diagnostic tool for multiplex nucleic acid detection, with an LOD down to 1 copy, high theoretical specificity, and halving of the amplification time compared to a nested qPCR assay.

scholarly journals Association of One-Step Nucleic Acid Amplification Detected Micrometastases with Tumour Biology and Adjuvant Chemotherapy

2017 ◽  
Vol 2017 ◽  
pp. 1-5 ◽  
Author(s):  
Ghaleb Goussous ◽  
Sadaf Jafferbhoy ◽  
Niamh Smyth ◽  
Lisette Hammond ◽  
Sankaran Narayanan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Adjuvant Chemotherapy ◽  
Sentinel Node ◽  
Survival Benefit ◽  
Nucleic Acid Amplification ◽  
Detection Rates ◽  
Tumour Biology ◽  
Number Of Patients ◽  
One Step ◽  
The Impact

One-step nucleic acid amplification (OSNA) is an intraoperative technique with a high sensitivity and specificity for sentinel node assessment. The aim of this study was to assess the impact of OSNA on micrometastases detection rates and use of adjuvant chemotherapy. A retrospective review of patients with sentinel node micrometastases over a five-year period was carried out and a comparison of micrometastases detection using OSNA and H&E techniques was made. Out of 1285 patients who underwent sentinel node (SLN) biopsy, 76 patients had micrometastases. Using H&E staining, 36 patients were detected with SLN micrometastases (9/year) in contrast to 40 patients in the OSNA year (40/year) (p<0.0001), demonstrating a fourfold increase with the use of OSNA. In the OSNA group, there was also a proportional increase in Grade III, triple-negative, ER-negative, and HER-2-positive tumours being diagnosed with micrometastases. Also on interactive PREDICT tool, the number of patients with a predicted 10-year survival benefit of more than 3% with adjuvant chemotherapy increased from 52 to 70 percent. OSNA has resulted in an increased detection rate of micrometastases especially in patients with aggressive tumour biology. This increased the number of patients who had a predicted survival benefit from adjuvant chemotherapy.

scholarly journals Development of a Portable SPR Sensor for Nucleic Acid Detection

Micromachines ◽  
2020 ◽  
Vol 11 (5) ◽  
pp. 526 ◽  
Author(s):  
Yafeng Huang ◽  
Lulu Zhang ◽  
Hao Zhang ◽  
Yichen Li ◽  
Luyao Liu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Incident Angle ◽  
Incident Light ◽  
High Sensitivity ◽  
Nucleic Acid Amplification ◽  
Nucleic Acid Hybridization ◽  
Nucleic Acid Detection ◽  
Scanning System ◽  
Label Free ◽  
Spr Sensor

Nucleic acid detection is of great significance in clinical diagnosis, environmental monitoring and food safety. Compared with the traditional nucleic acid amplification detection method, surface plasmon resonance (SPR) sensing technology has the advantages of being label-free, having simple operation, and providing real-time detection. However, the angle scanning system in many SPR angle modulation detection applications usually requires a high-resolution stepper motor and complex mechanical structure to adjust the angle. In this paper, a portable multi-angle scanning SPR sensor was designed. The sensor only uses one stepping motor to rotate a belt, and the belt pulls the mechanical linkages of incident light and reflected light to move in opposite directions for achieving the SPR angle scanning mode that keeps the incident angle and reflected angle equal. The sensor has an angle scanning accuracy of 0.002°, response sensitivity of 3.72 × 10−6 RIU (refractive index unit), and an angle scanning range of 30°–74°. The overall size of the system is only 480 mm × 150 mm × 180 mm. The portable SPR sensor was used to detect nucleic acid hybridization on a gold film chip modified with bovine serum albumin (BSA). The result revealed that the sensor had high sensitivity and fast response, and could successfully accomplish the hybridization detection of target DNA solution of 0.01 μmol/mL.

The increasing application of multiplex nucleic acid detection tests to the diagnosis of syndromic infections

2013 ◽  
Vol 142 (1) ◽  
pp. 1-11 ◽  
Author(s):  
J. GRAY ◽  
L. J. COUPLAND
Keyword(s):  
Nucleic Acid ◽  
Simultaneous Detection ◽  
Clinical Diagnostics ◽  
Nucleic Acid Amplification ◽  
Nucleic Acid Detection ◽  
Nucleic Acid Amplification Techniques ◽  
Infectious Gastroenteritis ◽  
The Usa ◽  
Acid Test

SUMMARYOn 14 January 2013, the US Food and Drug Administration (FDA) announced permission for a multiplex nucleic acid test, the xTAG®Gastrointestinal Pathogen Panel (GPP) (Luminex Corporation, USA), which simultaneously detects 11 common viral, bacterial and parasitic causes of infectious gastroenteritis, to be marketed in the USA. This announcement reflects the current move towards the development and commercialization of detection technologies based on nucleic acid amplification techniques for diagnosis of syndromic infections. We discuss the limitations and advantages of nucleic acid amplification techniques and the recent advances in Conformité Européene – in-vitrodiagnostic (CE-IVD)-approved multiplex real-time PCR kits for the simultaneous detection of multiple targets within the clinical diagnostics market.

scholarly journals Development of a Generic Microfluidic Device for Simultaneous Detection of Antibodies and Nucleic Acids in Oral Fluids

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Zongyuan Chen ◽  
William R. Abrams ◽  
Eran Geva ◽  
Claudia J. de Dood ◽  
Jesús M. González ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Microfluidic Device ◽  
Modular Design ◽  
Simultaneous Detection ◽  
Lateral Flow ◽  
Nucleic Acid Amplification ◽  
Antibody Detection ◽  
Confirmatory Test ◽  
Nucleic Acid Detection ◽  
Sample Processing

A prototype dual-path microfluidic device (Rheonix CARD) capable of performing simultaneously screening (antigen or antibody) and confirmatory (nucleic acid) detection of pathogens is described. The device fully integrates sample processing, antigen or antibody detection, and nucleic acid amplification and detection, demonstrating rapid and inexpensive “sample-to-result” diagnosis with performance comparable to benchtop analysis. For the chip design, a modular approach was followed allowing the optimization of individual steps in the sample processing process. This modular design provides great versatility accommodating different disease targets independently of the production method. In the detection module, a lateral flow (LF) protocol utilizing upconverting phosphor (UCP) reporters was employed. The nucleic acid (NA) module incorporates a generic microtube containing dry reagents. Lateral flow strips and PCR primers determine the target or disease that is diagnosed. Diagnosis of HIV infection was used as a model to investigate the simultaneous detection of both human antibodies against the virus and viral RNA. The serological result is available in less than 30 min, and the confirmation by RNA amplification takes another 60 min. This approach combines a core serological portable diagnostic with a nucleic acid-based confirmatory test.

The impact of One-Step Nucleic Acid Amplification (OSNA) on operative efficiency.

2014 ◽  
Vol 40 (5) ◽  
pp. 654
Author(s):  
Melissa Ley-Hui Tan ◽  
Lay In Lim ◽  
Ghaleb Goussous ◽  
Sankaran Narayanan ◽  
Robert Kirby ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Amplification ◽  
One Step ◽  
The Impact
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