scholarly journals Rapid Detection of Circulating Tumour DNA using Allele Specific Mini-Loop Mediated Isothermal Amplification

Author(s):  
Rebecca Allsopp ◽  
Georgios Alexandrou ◽  
Christofer Toumazou ◽  
Simak Ali ◽  
Charles Coombes ◽  
...  

Abstract Isothermal amplification is an emerging approach for non-invasive, rapid and cost-effective real-time monitoring of cancer specific mutations through circulating tumour DNA (ctDNA). This study demonstrates a compact allele specific (AS) loop mediated isothermal amplification (LAMP) strategy, termed ‘AS-Mini-LAMP’, modelled using wild type (WT) and mutation specific reactions targeting the estrogen receptor ESR1 c.1138G>C (p.E380Q) missense mutation. Allele selectivity, encoded at the 5’-end of the forward and backward inner primers (FIP and BIP) promotes enhanced selectivity upon self-hybridisation, loop formation and self-primed exponential amplification. Inclusion of unmodified self-stabilising (USS) primers aimed to reduce the likelihood of non-specific allele amplification through competitive inhibition and to enhance reaction velocity through an assisted strand displacement ‘swarm’ priming effect. The two assays were optimised using short synthetic WT and E380Q mutant DNA templates, and subsequently validated to a limit of detection of 500 mutant copies in under 25 minutes in ddPCR-confirmed positive (20.7% variant allele frequency) and negative patient plasma cfDNA samples. These results demonstrate the ability of AS-Mini-LAMP to achieve sensitive and selective amplification of actionable mutations present within plasma ctDNA.

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2187
Author(s):  
Paulina Rajko-Nenow ◽  
Emma L. A. Howson ◽  
Duncan Clark ◽  
Natasha Hilton ◽  
Aruna Ambagala ◽  
...  

Epizootic haemorragic disease (EHD) is an important disease of white-tailed deer and can cause a bluetongue-like illness in cattle. A definitive diagnosis of EHD relies on molecular assays such as real-time RT-qPCR or conventional PCR. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a cost-effective, specific, and sensitive technique that provides an alternative to RT-qPCR. We designed two sets of specific primers targeting segment-9 of the EHD virus genome to enable the detection of western and eastern topotypes, and evaluated their performance in singleplex and multiplex formats using cell culture isolates (n = 43), field specimens (n = 20), and a proficiency panel (n = 10). The limit of detection of the eastern and western RT-LAMP assays was estimated as ~24.36 CT and as ~29.37 CT in relation to real-time RT-qPCR, respectively, indicating a greater sensitivity of the western topotype singleplex RT-LAMP. The sensitivity of the western topotype RT-LAMP assay, relative to the RT-qPCR assay, was 72.2%, indicating that it could be theoretically used to detect viraemic cervines and bovines. For the first time, an RT-LAMP assay was developed for the rapid detection of the EHD virus that could be used as either a field test or high throughput screening tool in established laboratories to control the spread of EHD.


2021 ◽  
Vol 8 ◽  
Author(s):  
Alfredo Garcia-Venzor ◽  
Bertha Rueda-Zarazua ◽  
Eduardo Marquez-Garcia ◽  
Vilma Maldonado ◽  
Angelica Moncada-Morales ◽  
...  

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.


2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Xuzhi Zhang ◽  
Qianqian Yang ◽  
Qingli Zhang ◽  
Xiaoyu Jiang ◽  
Xiaochun Wang ◽  
...  

Abstract The cytochrome cd1-containing nitrite reductase, nirS, plays an important role in biological denitrification. Consequently, investigating the presence and abundance of nirS is a commonly used approach to understand the distribution and potential activity of denitrifying bacteria, in addition to denitrifier communities. Herein, a rapid method for detecting nirS gene with loop-mediated isothermal amplification (LAMP) was developed, using Pseudomonas aeruginosa PAO1 (P. aeruginosa PAO1) as model microorganism to optimize the assay. The LAMP assay relied on a set of four primers that were designed to recognize six target sequence sites, resulting in high target specificity. The limit of detection for the LAMP assay under optimized conditions was 1.87 pg/reaction of genomic DNA, which was an order of magnitude lower than that required by conventional PCR assays. Moreover, it was validated that P. aeruginosa PAO1 cells as well as genomic DNA could be directly used as template. Only 1 h was needed from the addition of bacterial cells to the reaction to the verification of amplification success. The nirS gene of P. aeruginosa PAO1 in spiked seawater samples could be detected with both DNA-template based LAMP assay and cell-template based LAMP assay, demonstrating the practicality of in-field use.


Author(s):  
Matthew A Lalli ◽  
Joshua S Langmade ◽  
Xuhua Chen ◽  
Catrina C Fronick ◽  
Christopher S Sawyer ◽  
...  

Abstract Background Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. Methods To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. Results The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44–104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. Conclusions Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.


Plant Disease ◽  
2019 ◽  
Vol 103 (12) ◽  
pp. 3142-3149
Author(s):  
Q. W. Wang ◽  
C. Q. Zhang

Trunk canker disease caused by Botryosphaeria dothidea with a prolonged latent infection phase poses a serious threat to Chinese hickory production. To further understand the epidemiological characteristics and develop reasonable management techniques, a quantitative loop-mediated isothermal amplification (q-LAMP) assay was developed to quantitatively monitor B. dothidea in hickory plants, water, and air samples. Specific primers were designed based on the different sites of the β-tubulin sequence between B. dothidea and other fungi commonly found on Chinese hickory. At the optimum reaction temperature of 65.9°C, this loop-mediated isothermal amplification (LAMP) assay can specifically distinguish B. dothidea from other tested fungi. The limit of detection of LAMP assays for B. dothidea was 0.001 ng/µl of pure genomic DNA and 10 spores per 1 ml of water. The q-LAMP assay enables rapid detection of B. dothidea within 60 min in hickory trunk, water in hickory forests, and spores captured on tapes. These results provide a powerful and convenient tool for monitoring B. dothidea, which could be applied widely in epidemiology, forecast, and management of tree canker disease.


2017 ◽  
Vol 07 (03) ◽  
pp. 042-048
Author(s):  
Gunimala Chakraborty ◽  
Indrani Karunasagar ◽  
Anirban Chakraborty

AbstractDelivery of quality healthcare in case of an infectious disease depends on how efficiently and how quickly the responsible pathogens are detected from the samples. Molecular methods can detect the presence of pathogens in a rapid and sensitive manner. Over the years, a number of such assays have been developed. However, these methods, although highly reliable and efficient, require use of expensive equipment, reagents, and trained personnel. Therefore, development of molecular assays that are simple, rapid, cost-effective, yet sensitive, is highly warranted to ensure efficient management or treatment strategies. Loop-mediated isothermal amplification (LAMP), a technique invented in the year 2000, is a novel method that amplifies DNA at isothermal conditions. Since its invention, this technique has been one of the most extensively used molecular diagnostic tools in the field of diagnostics offering rapid, accurate and cost-effective diagnosis of infectious diseases. Using the LAMP principle, many commercial kits have been developed in the last decade for a variety of human pathogens including bacteria, viruses and parasites. Currently LAMP assay is being considered as an effective diagnostic tool for use in developing countries because of its simple working protocol, allowing even an onsite application. The focus of this review is to describe the salient features of this technique the current status of development of LAMP assays with an emphasis on the pathogens of clinical significance.


2021 ◽  
Author(s):  
Everardo González-González ◽  
Itzel Montserrat Lara-Mayorga ◽  
Iram Pablo Rodríguez-Sánchez ◽  
Yu Shrike Zhang ◽  
Sergio O. Martínez-Chapa ◽  
...  

Colorimetric LAMP for COVID-19 intensified diagnostics: a simple and quantitative method comparable in diagnostic performance to RT-qPCR.


Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 972 ◽  
Author(s):  
Mohammed A. Rohaim ◽  
Emily Clayton ◽  
Irem Sahin ◽  
Julianne Vilela ◽  
Manar E. Khalifa ◽  
...  

Until vaccines and effective therapeutics become available, the practical solution to transit safely out of the current coronavirus disease 19 (CoVID-19) lockdown may include the implementation of an effective testing, tracing and tracking system. However, this requires a reliable and clinically validated diagnostic platform for the sensitive and specific identification of SARS-CoV-2. Here, we report on the development of a de novo, high-resolution and comparative genomics guided reverse-transcribed loop-mediated isothermal amplification (LAMP) assay. To further enhance the assay performance and to remove any subjectivity associated with operator interpretation of results, we engineered a novel hand-held smart diagnostic device. The robust diagnostic device was further furnished with automated image acquisition and processing algorithms and the collated data was processed through artificial intelligence (AI) pipelines to further reduce the assay run time and the subjectivity of the colorimetric LAMP detection. This advanced AI algorithm-implemented LAMP (ai-LAMP) assay, targeting the RNA-dependent RNA polymerase gene, showed high analytical sensitivity and specificity for SARS-CoV-2. A total of ~200 coronavirus disease (CoVID-19)-suspected NHS patient samples were tested using the platform and it was shown to be reliable, highly specific and significantly more sensitive than the current gold standard qRT-PCR. Therefore, this system could provide an efficient and cost-effective platform to detect SARS-CoV-2 in resource-limited laboratories.


2019 ◽  
Vol 47 (21) ◽  
pp. e141-e141 ◽  
Author(s):  
Antoine Daunay ◽  
Alex Duval ◽  
Laura G Baudrin ◽  
Olivier Buhard ◽  
Victor Renault ◽  
...  

Abstract Microsatellites are polymorphic short tandem repeats of 1–6 nucleotides ubiquitously present in the genome that are extensively used in living organisms as genetic markers and in oncology to detect microsatellite instability (MSI). While the standard analysis method of microsatellites is based on PCR followed by capillary electrophoresis, it generates undesirable frameshift products known as ‘stutter peaks’ caused by the polymerase slippage that can greatly complicate the analysis and interpretation of the data. Here we present an easy multiplexable approach replacing PCR that is based on low temperature isothermal amplification using recombinase polymerase amplification (LT-RPA) that drastically reduces and sometimes completely abolishes the formation of stutter artifacts, thus greatly simplifying the calling of the alleles. Using HT17, a mononucleotide DNA repeat that was previously proposed as an optimal marker to detect MSI in tumor DNA, we showed that LT-RPA improves the limit of detection of MSI compared to PCR up to four times, notably for small deletions, and simplifies the identification of the mutant alleles. It was successfully applied to clinical colorectal cancer samples and enabled detection of MSI. This easy-to-handle, rapid and cost-effective approach may deeply improve the analysis of microsatellites in several biological and clinical applications.


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