https://link.springer.com/content/pdf/10.1007%2Fs10156-009-0669-9.pdf

Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases

Loop mediated isothermal amplification (LAMP) is a powerful innovative gene amplification technique emerging as a simple rapid diagnostic tool for early detection and identification of microbial diseases. The whole procedure is very simple and rapid wherein the amplification can be completed in less than 1 h under isothermal conditions employing a set of six specially designed primers spanning eight distinct sequences of a target gene, by incubating all the reagents in a single tube. Gene amplification products can be detected by agarose gel electrophoresis as well as by real-time monitoring in an inexpensive turbidimeter. Gene copy number can also be quantified with the help of a standard curve generated from different concentrations of gene copy number plotted against time of positivity with the help of a real-time turbidimeter. Alternatively, gene amplification can be visualised by the naked eye either as turbidity or in the form of a colour change when SYBR Green I, a fluorescent dsDNA intercalating dye, is employed. LAMP does not require a thermal cycler and can be performed simply with a heating block and/or water bath. Considering the advantages of rapid amplification, simple operation and easy detection, LAMP has potential applications for clinical diagnosis as well as surveillance of infectious diseases in developing countries without requiring sophisticated equipment or skilled personnel. Copyright © 2008 John Wiley & Sons, Ltd.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/rmv.593

Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases.

Nucleic acid amplification technologies are used in the field of molecular biology and recombinant DNA technologies. These techniques are used as leading methods in detecting and analyzing a small quantity of nucleic acids. The polymerase chain reaction (PCR) is the most widely used method for DNA amplification for detection and identification of infectious diseases, genetic disorders and other research purposes. However, it requires a thermocycling machine to separate two DNA strands and then amplify the required fragment. Novel developments in molecular biology of DNA synthesis in vivo demonstrate the possibility of amplifying DNA in isothermal conditions without the need of a thermocycling apparatus. DNA polymerase replicates DNA with the aid of various accessory proteins. Recent identification of these proteins has enabled development of new in vitro isothermal DNA amplification methods, mimicking these in vivo mechanisms. There are several types of isothermal nucleic acid amplification methods such as transcription mediated amplification, nucleic acid sequence-based amplification, signal mediated amplification of RNA technology, strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification of DNA, isothermal multiple displacement amplification, helicase-dependent amplification, single primer isothermal amplification, and circular helicase-dependent amplification. In this article, we review these isothermal nucleic acid amplification technologies and their applications in molecular biological studies.

Nucleic Acid Isothermal Amplification Technologies—A Review

West Nile virus (WNV) causes epidemics of febrile illness, meningitis, encephalitis, and flaccid paralysis. Since it was first detected in New York City in 1999, and through 2004, 16,000 WNV disease cases have been reported in the United States. Over the past 5 years, research on WNV disease has expanded rapidly. This review highlights new information regarding the virology, clinical manifestations, and pathology of WNV disease, which will provide a new platform for further research into diagnosis, treatment, and possible prevention of WNV through vaccination.

/pdf/virology-pathology-and-clinical-manifestations-of-west-nile-37cv9vkhpc.pdf

Virology, pathology, and clinical manifestations of West Nile virus disease.

Purpose: Detection of sentinel lymph node (SLN) metastasis in breast cancer patients has conventionally been determined by intraoperative histopathologic examination of frozen sections followed by definitive postoperative examination of permanent sections. The purpose of this study is to develop a more efficient method for intraoperative detection of lymph node metastasis. Experimental Design: Cutoff values to distinguish macrometastasis, micrometastasis, and nonmetastasis were determined by measuring cytokeratin 19 (CK19) mRNA in histopathologically positive and negative lymph nodes using one-step nucleic acid amplification (OSNA). In an intraoperative clinical study involving six facilities, 325 lymph nodes (101 patients), including 81 SLNs, were divided into four blocks. Alternate blocks were used for the OSNA assay with CK19 mRNA, and the remaining blocks were used for H&E and CK19 immunohistochemistry–based three-level histopathologic examination. The results from the two methods were then compared. Results: We established CK19 mRNA cutoff values of 2.5 × 10 2 and 5 × 10 3 copies/μL. In the clinical study, an overall concordance rate between the OSNA assay and the three-level histopathology was 98.2%. Similar results were obtained with 81 SLNs. The OSNA assay discriminated macrometastasis from micrometastasis. No false positive was observed in the OSNA assay of 144 histopathologically negative lymph nodes from pN0 patients, indicating an extremely low false positive for the OSNA assay. Conclusion: The OSNA assay of half of a lymph node provided results similar to those of three-level histopathology. Clinical results indicate that the OSNA assay provides a useful intraoperative detection method of lymph node metastasis in breast cancer patients.

https://clincancerres.aacrjournals.org/content/clincanres/13/16/4807.full.pdf

One-step Nucleic Acid Amplification for Intraoperative Detection of Lymph Node Metastasis in Breast Cancer Patients

A one-step, single tube, real-time accelerated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting the envelope gene of West Nile (WN) virus. The RT-LAMP assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, efficiency, and rapidity under isothermal conditions with a set of six specially designed primers that recognize eight distinct sequences of the target. The whole procedure is very simple and rapid, and amplification can be obtained in less than 1 h by incubating all of the reagents in a single tube with reverse transcriptase and Bst DNA polymerase at 63°C. Detection of gene amplification could be accomplished by agarose gel electrophoresis, as well as by real-time monitoring in an inexpensive turbidimeter. When the sensitivity of the RT-LAMP assay was compared to that of conventional RT-PCR, it was found that the RT-LAMP assay demonstrated 10-fold higher sensitivity compared to RT-PCR, with a detection limit of 0.1 PFU of virus. By using real-time monitoring, 104 PFU of virus could be detected in as little as 17 min. The specificity of the RT-LAMP assay was validated by the absence of any cross-reaction with other, closely related, members of the Flavivirus group, followed by restriction digestion and nucleotide sequencing of the amplified product. These results indicate that the RT-LAMP assay is extremely rapid, cost-effective, highly sensitive, and specific and has potential usefulness for rapid, comprehensive WN virus surveillance along with virus isolation and/or serology.

Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of West Nile Virus

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Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of West Nile Virus