scholarly journals Simple and rapid detection of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans by loop-mediated isothermal amplification assay

2018 ◽  
Vol 17 (3) ◽  
pp. 402-410 ◽  
Author(s):  
Nurul Izzati Hamzan ◽  
Fatin Hazwani Fauzi ◽  
Haslina Taib ◽  
Suharni Mohamad
Keyword(s):  
Porphyromonas Gingivalis ◽  
Rapid Detection ◽  
Detection Rate ◽  
Medical Science ◽  
Lamp Assay ◽  
Dna Amplification ◽  
Cost Effective ◽  
Aggregatibacter Actinomycetemcomitans ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification

Background: Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are two main causative agents associated with periodontitis, an inflammatory reaction of tissues around the teeth. The aim of this study was to develop and evaluate the loop-mediated isothermal amplification (LAMP) assay for simple and rapid detection of P. gingivalis and A. actinomycetemcomitans.Methods: A total of ten subgingival plaque and saliva samples were evaluated to detect the presence of both bacteria by LAMP and PCR assays. Two sets of six primers each were designed to amplify pepO and dam gene. The LAMP assay was carried out using a Loopamp DNA amplification kit in 25 μl volumes. The reaction mixture was incubated at 65oC for 60 minutes and terminated at 80oC for 5 minutes in heating block. The amplification reactions were visualized using naked eye detection and by agarose gel electrophoresis. The sensitivity of the LAMP assay was investigated ranging from 10 μg to 100fg of P. gingivalis(ATCC 33327) and A. actinomycetemcomitans (ATCC 33384).Results: The lowest detection limit of both LAMP and PCR methods were 1 ng and 10 ng of DNA, respectively. When crude template of subgingival plaques were used, P. gingivalisand A. actinomycetemcomitans were tested80% (8/10) and 60% (6/10) positive respectively through LAMP detection. Whereas by PCR, P. gingivaliswas tested 40% (4/10) positive and no significant detection rate for A. actinomycetemcomitans. When a crude template of saliva was used, P. gingivalisand A. actinomycetemcomitans were tested 70% (7/10) and 30% (3/10) positive respectively through LAMP detection. Whereas, when using PCR, there was no significant detection rate for P. gingivalisand A. actinomycetemcomitans.Conclusion: The LAMP assay using a crude template offers greater advantage as it is simple, rapid and cost-effective to detect periodontal pathogens.Bangladesh Journal of Medical Science Vol.17(3) 2018 p.402-410

scholarly journals Development of a Novel Loop Mediated Isothermal Amplification Assay (LAMP) for the Rapid Detection of Epizootic Haemorrhagic Disease Virus

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2187
Author(s):  
Paulina Rajko-Nenow ◽  
Emma L. A. Howson ◽  
Duncan Clark ◽  
Natasha Hilton ◽  
Aruna Ambagala ◽  
...  
Keyword(s):  
Real Time ◽  
Rapid Detection ◽  
High Throughput Screening ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Cost Effective ◽  
Virus Genome ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Haemorrhagic Disease

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.

scholarly journals Development and validation of a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in patient samples from Brazil

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Severino Jefferson Ribeiro da Silva ◽  
Keith Pardee ◽  
Udeni B. R. Balasuriya ◽  
Lindomar Pena
Keyword(s):  
Reverse Transcription ◽  
Rapid Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Serum Samples ◽  
Low Resource ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

AbstractWe have previously developed and validated a one-step assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of the Zika virus (ZIKV) from mosquito samples. Patient diagnosis of ZIKV is currently carried out in centralized laboratories using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which, while the gold standard molecular method, has several drawbacks for use in remote and low-resource settings, such as high cost and the need of specialized equipment. Point-of-care (POC) diagnostic platforms have the potential to overcome these limitations, especially in low-resource countries where ZIKV is endemic. With this in mind, here we optimized and validated our RT-LAMP assay for rapid detection of ZIKV from patient samples. We found that the assay detected ZIKV from diverse sample types (serum, urine, saliva, and semen) in as little as 20 min, without RNA extraction. The RT-LAMP assay was highly specific and up to 100 times more sensitive than RT-qPCR. We then validated the assay using 100 patient serum samples collected from suspected cases of arbovirus infection in the state of Pernambuco, which was at the epicenter of the last Zika epidemic. Analysis of the results, in comparison to RT-qPCR, found that the ZIKV RT-LAMP assay provided sensitivity of 100%, specificity of 93.75%, and an overall accuracy of 95.00%. Taken together, the RT-LAMP assay provides a straightforward and inexpensive alternative for the diagnosis of ZIKV from patients and has the potential to increase diagnostic capacity in ZIKV-affected areas, particularly in low and middle-income countries.

scholarly journals Loop-mediated isothermal amplification (LAMP) : A rapid molecular diagnosis technique for detection of human pathogens

2017 ◽  
Vol 07 (03) ◽  
pp. 042-048
Author(s):  
Gunimala Chakraborty ◽  
Indrani Karunasagar ◽  
Anirban Chakraborty
Keyword(s):  
Treatment Strategies ◽  
Lamp Assay ◽  
Cost Effective ◽  
Isothermal Amplification ◽  
Current Status ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Human Pathogens ◽  
Quality Healthcare ◽  
Loop Mediated Isothermal Amplification

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.

scholarly journals Artificial Intelligence-Assisted Loop Mediated Isothermal Amplification (AI-LAMP) for Rapid Detection of SARS-CoV-2

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 972 ◽  
Author(s):  
Mohammed A. Rohaim ◽  
Emily Clayton ◽  
Irem Sahin ◽  
Julianne Vilela ◽  
Manar E. Khalifa ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
De Novo ◽  
Tracking System ◽  
Lamp Assay ◽  
Cost Effective ◽  
Isothermal Amplification ◽  
Analytical Sensitivity ◽  
Diagnostic Device ◽  
Loop Mediated Isothermal Amplification ◽  
Resource Limited

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.

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