A novel method to control carryover contamination in isothermal nucleic acid amplification

2017 ◽  
Vol 53 (77) ◽  
pp. 10696-10699 ◽  
Author(s):  
Cuiping Ma ◽  
Fuxin Wang ◽  
Xiudan Wang ◽  
Lingzhi Han ◽  
Hao Jing ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Restriction Endonuclease ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Isothermal Nucleic Acid Amplification ◽  
Recognition Sites ◽  
Novel Method

We developed a novel method to control carryover contamination in loop-mediated isothermal amplification (LAMP) by primer engineering to carry recognition sites for a restriction endonuclease, providing a robust ability to eliminate carryover contaminants.

scholarly journals High-Surety Isothermal Amplification and Detection of SARS-CoV-2

mSphere ◽  
2021 ◽  
Vol 6 (3) ◽  
Author(s):  
Sanchita Bhadra ◽  
Timothy E. Riedel ◽  
Simren Lakhotia ◽  
Nicholas D. Tran ◽  
Andrew D. Ellington
Keyword(s):  
Nucleic Acid ◽  
False Negative ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Boolean Logic ◽  
Nucleic Acid Amplification ◽  
Rapid Diagnostics ◽  
One Pot ◽  
Loop Mediated Isothermal Amplification ◽  
Isothermal Nucleic Acid Amplification

ABSTRACT Isothermal nucleic acid amplification tests (iNATs), such as loop-mediated isothermal amplification (LAMP), are good alternatives to PCR-based amplification assays, especially for point-of-care and low-resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can often generate spurious amplicons, especially in the absence of target sequences, resulting in false-positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets and can accumulate mutations that will lead to inefficient primer binding and thus false-negative results. Multiplex assays targeting different regions of the analyte and logical signal readout using sequence-specific probes can help to reduce both false negatives and false positives. Here, we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on a non-sequence-specific readout into individual and multiplex one-pot assays that can be visually read using sequence-specific oligonucleotide strand exchange (OSD) probes. We describe both fluorescence-based and Boolean logic-gated colorimetric lateral flow readout methods and demonstrate detection of SARS-CoV-2 virions in crude human saliva. IMPORTANCE One of the key approaches to treatment and control of infectious diseases, such as COVID-19, is accurate and rapid diagnostics that is widely deployable in a timely and scalable manner. To achieve this, it is essential to go beyond the traditional gold standard of quantitative PCR (qPCR) that is often faced with difficulties in scaling due to the complexity of infrastructure and human resource requirements. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), have been long pursued as ideal, low-tech alternatives for rapid, portable testing. However, isothermal approaches often suffer from false signals due to employment of nonspecific readout methods. We describe general principles for rapidly converting nonspecifically read LAMP assays into assays that are read in a sequence-specific manner by using oligonucleotide strand displacement (OSD) probes. We also demonstrate that inclusion of OSD probes in LAMP assays maintains the simplicity of one-pot assays and a visual yes/no readout by using fluorescence or colorimetric lateral-flow dipsticks while providing accurate sequence-specific readout and the ability to logically query multiplex amplicons for redundancy or copresence. These principles not only yielded high-surety isothermal assays for SARS-CoV-2 but might also aid in the design of more sophisticated molecular assays for other analytes.

Long-term dry storage of enzyme-based reagents for isothermal nucleic acid amplification in a porous matrix for use in point-of-care diagnostic devices

The Analyst ◽  
2020 ◽  
Vol 145 (21) ◽  
pp. 6875-6886 ◽  
Author(s):  
Sujatha Kumar ◽  
Ryan Gallagher ◽  
Josh Bishop ◽  
Enos Kline ◽  
Joshua Buser ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Glass Fiber ◽  
Point Of Care ◽  
Porous Matrix ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Isothermal Nucleic Acid Amplification ◽  
Dry Storage

Long-term dry storage of enzyme-based isothermal amplification reagents in glass fiber porous matrix for use in point-of-care devices.

scholarly journals Loop-Mediated Isothermal Amplification (LAMP) for Detection of Various Organisms: A Review

2017 ◽  
Vol 11 (2) ◽  
pp. 20-27
Author(s):  
Arifa Akram
Keyword(s):  
Nucleic Acid ◽  
High Specificity ◽  
Disease Diagnosis ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Lamp Method ◽  
Isolation And Identification ◽  
Strand Displacement Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Prophylactic Measures

Disease diagnosis is important for implementation of proper therapeutic and prophylactic measures. Traditionally, disease diagnosis was depends upon isolation and identification of the causative organisms. This was followed by serology and after that molecular method. Molecular tests are valuable when early diagnosis is important. For this purpose, nucleic acid amplification (PCR, nucleic acid sequence-based amplification, self-sustained sequence replication, strand displacement amplification) is one of the most valuable tools not only for the diagnosis of infectious diseases but also used in advanced level research. The Loop-Mediated Isothermal Amplification (LAMP) is a unique nucleic acid amplification technique for diagnosis of various pathogens introduced at 2000 by Notomi and his colleagues which is simple, easy, rapid and cost effective when compared to PCR due to its high specificity, sensitivity, and rapidity. It uses a set of six primers and a DNA polymerase with stranddisplacement activity. Major advantage of LAMP method is its cost-effectiveness as it can be done simply by using waterbath or heating block. Bangladesh J Med Microbiol 2017; 11 (2): 20-27

DNA-templated copper nanoclusters obtained via TdT isothermal nucleic acid amplification for mercury(ii) assay

2019 ◽  
Vol 11 (32) ◽  
pp. 4165-4172 ◽  
Author(s):  
Jing-Lin He ◽  
Xing-Xing Wang ◽  
Ting-Ting Mei ◽  
Ling Wu ◽  
Ju-Lan Zeng ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Fluorescence Sensor ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Copper Nanoclusters ◽  
Isothermal Nucleic Acid Amplification

The Hg2+ fluorescence sensor based on TdT isothermal amplification DNA templated CuNCs was successfully constructed.

A novel exonuclease-assisted isothermal nucleic acid amplification with ultrahigh specificity mediated by full-length Bst DNA polymerase

2018 ◽  
Vol 54 (75) ◽  
pp. 10562-10565 ◽  
Author(s):  
Xin Ye ◽  
Yang Li ◽  
Lijuan Wang ◽  
Xueen Fang ◽  
Jilie Kong
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Dna Polymerase ◽  
Isothermal Amplification ◽  
Full Length ◽  
Nucleic Acid Amplification ◽  
Isothermal Nucleic Acid Amplification

A novel exonuclease-assisted isothermal amplification to amplify and determine nucleic acids very sensitively and with ultrahigh specificity.

scholarly journals Clinical profiling and use of loop-mediated isothermal amplification assay for rapid detection of Mycobacterium tuberculosis from sputum

1970 ◽  
Vol 7 (2) ◽  
pp. 109-114 ◽  
Author(s):  
A Poudel ◽  
BD Pandey ◽  
B Lekhak ◽  
B Rijal ◽  
BR Sapkota ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nucleic Acid ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Common Symptom ◽  
Bcg Vaccination ◽  
Loop Mediated Isothermal Amplification ◽  
16S Rna ◽  
Clinical Profiling

Background: Tuberculosis is a global health problem and the situation is worsening with newer incidences of drug resistance and HIV association. Diagnosis of tuberculosis can be done by many methods and test, culture of sputum being the ideal one. Nucleic acid amplification (NAA) assay are more time efficient one, that amplify and detect specific nucleic acid sequences allows rapid, sensitive and specific detection of M. tuberculosis in sputum samples. Objectives: The present study intends to compile the clinical presentations of the pulmonary tuberculosis (PTB) patients and to evaluate the efficacy of in-house loop-mediated isothermal amplification (LAMP) in detecting Mycobacterium tuberculosis in sputum samples by comparing with microscopy and culture. Materials and methods: Two hundred two sputum samples were collected from 202 patients at National Tuberculosis Center, Bhaktapur, Nepal. Complete clinical profiling, epidemiological data and record on BCG vaccination were noted and the samples were subjected for microscopy, culture and in-house LAMP with six primers specific for 16S RNA gene of Mycobacterium tuberculosis. Result: Of the 176 cases of clinical profiling, productive cough was most common symptom in 147 (83.52%), followed by chest pain 136 (77.27%), fever 133 (75.56%) and haemoptysis 61 (34.66%). There was a statistically significant association between BCG vaccination and development of TB (Χ2=5.33, P=0.02). Of 202 cases, 115 (56.93%) were chest X-ray positive, 101(50%) were direct smear-positive and 100 (49.51%) were culture positive. LAMP had a sensitivity of 97% and specificity of 94.12% while comparing with culture. In addition, its sensitivity and specificity were 91.09% and 89.11% respectively with reference to microscopy. Conclusion: As in our previous study, overall, the result of present study further confirms that the in-house LAMP is a simple, rapid, sensitive and specific DNA amplification technique for PTB diagnosis. Because of rapidity of microscopy and specificity of culture, in-house LAMP assay can be used as a very powerful and useful supplementary tool with complete clinical profiling of the patients for rapid diagnosis of TB in both AFB-positive and negative cases who are suspected as PTB in disease endemic country like Nepal. Key words: clinical profiling; Sputum; DNA; LAMP; M. tuberculosis; Nepal DOI: 10.3126/kumj.v7i2.2701 Kathmandu University Medical Journal (2009) Vol.7, No.2 Issue 26, 109-114

scholarly journals Strategies for Isothermal Amplification of Nucleic Acids: are they Ready to be Applied in Point of Care Diagnosis of Mycosis?

2020 ◽  
Vol 11 (3) ◽  
pp. 10559-10571
Keyword(s):  
Nucleic Acid ◽  
Clinical Laboratory ◽  
Multiple Displacement Amplification ◽  
Rolling Circle Amplification ◽  
High Sensitivity ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Rolling Circle ◽  
Isothermal Nucleic Acid Amplification

The early detection of invasive fungal infection (IFD) is significant in order to decrease mortality in susceptible patients. There is, therefore, a need for sensitive and specific fungal species detection assays in a clinical laboratory for early targeted therapy. The isothermal amplification method may be useful for the screening of fungal isolates, especially in resource-poor settings. Therefore, our aim was to review the isothermal nucleic acid amplification methods and their applications in fungal pathogen detection. Out of 50 reported studies, 28, 12, 6, 2, and 2 studies used the isothermal-based assays of a loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), multiple displacement amplification (MDA) and polymerase Spiral Reaction (PSR), respectively. Thirty-two studies used clinical samples, 18 pure culture, and four environmental samples. The diagnostic accuracy of isothermal nucleic acid amplification testing for pathogenic fungal was reported as high (sensitivity 0.89–1.0 and specificity 0.63–1.0) in all studies irrespective of the sample tested. Although the isothermal-based assays showed high sensitivity and specificity in reported studies, it is still poorer than that of PCR assays. However, improving the assay to make it simpler, more effective, and inexpensive compared with newer PCR methods are still needed.

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