scholarly journals A Stoichiometric and Pseudo Kinetic Model of Loop Mediated Isothermal Amplification

2020 ◽  
Author(s):  
Navjot Kaur ◽  
Nikhil Thota ◽  
Bhushan Toley
Keyword(s):  
Kinetic Model ◽  
Reaction Pathway ◽  
Reaction Network ◽  
Dna Amplification ◽  
Lamp Reaction ◽  
Isothermal Amplification ◽  
Reaction Products ◽  
Theoretical Understanding ◽  
Loop Mediated Isothermal Amplification ◽  
Commercial Applications

<p>Loop mediated isothermal amplification (LAMP) is one of the most popular isothermal DNA amplification techniques for research and commercial applications. The LAMP mechanism is powered by strategic primer design and a strand displacement polymerase, generating products that fold over, creating loops. LAMP leads to generation of products of increasing length over time. These products containing multiple loops are conventionally called cauliflower structures. Existing literature on LAMP provides extremely limited understanding of progression of cascades of reactions involved in the reaction and it is believed that cauliflower structures of increasing length constitute a majority of the product formed in LAMP. This study presents a first of its kind stoichiometric and pseudo kinetic model to comprehend LAMP reactions in deeper depth by (i) classifying LAMP reaction products into uniquely identifiable categories, (ii) generating a condensed reaction network to depict millions of interconnected reactions occurring during LAMP, and (iii) elucidating the pathways for amplicon generation. Despite the inherent limitations of conventional stoichiometric modelling for polymerization type reactions (the network rapidly becomes too large and intractable), our model provides new theoretical understanding of the LAMP reaction pathway. The model shows that while longer length products are formed, it is the smaller length recycle amplicons that contribute more towards the exponential increase in the amount of double stranded DNA. Prediction of concentration of different types of LAMP amplicons will also contribute substantially towards informing design of probe-based assays. </p>

scholarly journals A Stoichiometric and Pseudo Kinetic Model of Loop Mediated Isothermal Amplification

2020 ◽  
Author(s):  
Navjot Kaur ◽  
Nikhil Thota ◽  
Bhushan Toley
Keyword(s):  
Kinetic Model ◽  
Reaction Pathway ◽  
Reaction Network ◽  
Dna Amplification ◽  
Lamp Reaction ◽  
Isothermal Amplification ◽  
Reaction Products ◽  
Theoretical Understanding ◽  
Loop Mediated Isothermal Amplification ◽  
Commercial Applications

<p>Loop mediated isothermal amplification (LAMP) is one of the most popular isothermal DNA amplification techniques for research and commercial applications. The LAMP mechanism is powered by strategic primer design and a strand displacement polymerase, generating products that fold over, creating loops. LAMP leads to generation of products of increasing length over time. These products containing multiple loops are conventionally called cauliflower structures. Existing literature on LAMP provides extremely limited understanding of progression of cascades of reactions involved in the reaction and it is believed that cauliflower structures of increasing length constitute a majority of the product formed in LAMP. This study presents a first of its kind stoichiometric and pseudo kinetic model to comprehend LAMP reactions in deeper depth by (i) classifying LAMP reaction products into uniquely identifiable categories, (ii) generating a condensed reaction network to depict millions of interconnected reactions occurring during LAMP, and (iii) elucidating the pathways for amplicon generation. Despite the inherent limitations of conventional stoichiometric modelling for polymerization type reactions (the network rapidly becomes too large and intractable), our model provides new theoretical understanding of the LAMP reaction pathway. The model shows that while longer length products are formed, it is the smaller length recycle amplicons that contribute more towards the exponential increase in the amount of double stranded DNA. Prediction of concentration of different types of LAMP amplicons will also contribute substantially towards informing design of probe-based assays. </p>

scholarly journals Reverse Transcription Loop-Mediated Isothermal Amplification for the Detection of Porcine Reproductive and Respiratory Syndrome Virus

2009 ◽  
Vol 21 (3) ◽  
pp. 350-354 ◽  
Author(s):  
Albert Rovira ◽  
Juan Abrahante ◽  
Michael Murtaugh ◽  
Muñoz-Zanzi Claudia
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Restriction Analysis ◽  
Dna Amplification ◽  
Lamp Reaction ◽  
Isothermal Amplification ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
Serum Samples ◽  
Loop Mediated Isothermal Amplification

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine. The objective of the current study is to investigate the feasibility of using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of PRRSV. The RT-LAMP is a recently described DNA amplification technique reported to be simple, inexpensive, fast, and accurate. The RT-LAMP reaction was set up using 2 sets of primers that were designed to detect North American and European strains of PRRSV and performed successfully in a simple heat block. The specificity of the amplified product was demonstrated by restriction analysis. The RT-LAMP was able to detect 5 different PRRSV isolates. However, the limit of detection ranged between 10 2 and 10 4 50% tissue culture infective dose/ml. The RT-LAMP was further evaluated using serum samples from animals of known infection status. The ability of RT-LAMP to detect PRRSV in serum from acutely infected animals was evaluated with 114 serum samples from 18 experimentally inoculated boars. Forty-nine of these samples tested positive by RT-LAMP, while 94 were positive by reverse transcription polymerase chain reaction (RT-PCR). The diagnostic specificity, evaluated with 100 known negative serum samples, was estimated as 99%. The feasibility of RT-LAMP to detect PRRSV was demonstrated in the current study. The RT-LAMP reaction could be performed in just 1 hr with a simple and inexpensive heat block. However, the sensitivity of this technique was significantly lower than that of RT-PCR.

scholarly journals RNase H-dependent amplification improves the accuracy of rolling circle amplification combined with loop-mediated isothermal amplification (RCA-LAMP)

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11851
Author(s):  
Takema Hasegawa ◽  
Diana Hapsari ◽  
Hitoshi Iwahashi
Keyword(s):  
Detection Limit ◽  
Rolling Circle Amplification ◽  
Dna Amplification ◽  
Rnase H ◽  
Isothermal Amplification ◽  
Rolling Circle ◽  
Loop Mediated Isothermal Amplification ◽  
Specific Amplification ◽  
Negative Condition ◽  
Rnase H2

The hybrid method upon combining rolling circle amplification and loop-mediated isothermal amplification (RCA-LAMP) was developed to quantify very small amount of different type of RNAs, such as miRNAs. RCA-LAMP can help detect short sequences through padlock probe (PLP) circularization and exhibit powerful DNA amplification. However, one of the factors that determines the detection limit of RCA-LAMP is non-specific amplification. In this study, we improved the accuracy of RCA-LAMP through applying RNase H-dependent PCR (rhPCR) technology. In this method, the non-specific amplification was suppressed by using the rh primer, which is designed through blocking the modification at the 3′end to stop DNA polymerase reaction and replacing the 6th DNA molecule from the end with RNA using RNase H2 enzyme. Traditional RCA-LAMP amplified the non-specific amplicons from linear PLP without a targeting reaction, while RCA-LAMP with rh primer and RNase H2 suppressed the non-specific amplification. Conversely, we identified the risk posed upon conducting PLP cyclization reaction using Splint R ligase in the RNA-targeting step that occurred even in the RNA-negative condition, which is another factor determining the detection limit of RCA-LAMP. Therefore, this study contributes in improving the accuracy of RNA quantification using RCA-LAMP.

scholarly journals Polyethylene Glycol Accelerates Loop-mediated Isothermal Amplification (LAMP) Reaction

2013 ◽  
Vol 133 (10) ◽  
pp. 1121-1126 ◽  
Author(s):  
Kenta Nose ◽  
Kentaro Nagamine ◽  
Junichi Tokuda ◽  
Junichi Takino ◽  
Takamitsu Hori
Keyword(s):  
Polyethylene Glycol ◽  
Lamp Reaction ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification

scholarly journals Reverse Transcription Loop-Mediated Isothermal Amplification of DNA for Detection of Potato virus Y

Plant Disease ◽  
2005 ◽  
Vol 89 (6) ◽  
pp. 605-610 ◽  
Author(s):  
Xianzhou Nie
Keyword(s):  
Potato Virus ◽  
Reverse Transcription ◽  
Potato Virus Y ◽  
Dna Amplification ◽  
Isothermal Amplification ◽  
Rt Pcr ◽  
Potato Leaf ◽  
Time Saving ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

A reverse transcription loop-mediated isothermal amplification of DNA (RT-LAMP) for detection of Potato virus Y (PVY) was developed. In this procedure, a set of four primers matching a total of six sequences of the coat protein (CP) gene of PVY were designed in such a way that a loop could be formed and elongated during DNA amplification. Using PVY CP complementary DNA clones as templates, the LAMP reaction was optimized by adjusting the concentrations of MgSO4, dNTPs, and Bst DNA polymerase. The effects of fragment length of target DNA on LAMP also were investigated. Two-step and one-step RT-LAMPs were performed using RNA extracts of various PVY cultures, and the results were correlated with two-step reverse transcription polymerase chain reaction (RT-PCR) for detection of PVY. Further, the turbidity caused by precipitation of magnesium pyrophosphate formed in positive RT-LAMP reactions was used to measure the amplification by utilizing a time-saving spectrophotometric method. The one-step RT-LAMP-turbidity method gave results comparable with the two-step RT-PCR method for detection of PVY from potato leaf and tuber samples. Of the total 240 samples, 234 were diagnosed similarly by both methods.

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

Rapid Detection of Mycobacterium tuberculosis Complex by Loop-Mediated Isothermal Amplification Combined with Chemosensor

2013 ◽  
Vol 749 ◽  
pp. 449-452
Author(s):  
De Guo Wang
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Amplification ◽  
Raw Milk ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Milk Samples ◽  
Assay Time ◽  
Low Levels ◽  
Rapid Amplification

Loop-mediated isothermal amplification (LAMP) allowed rapid amplification of nucleic acids under isothermal conditions. It can be combined with a chemosensor for much more efficient, field-friendly detection of Mycobacterium tuberculosis complex. In this report, LAMP was performed at 65 °C for 10 min, followed by a rapid reaction of DNA amplification by-product, pyrophosphate ion, with chemosensor resulted in red disappearance. The detection limit of Mycobacterium tuberculosis complex by LAMP-Chemosensor was 3-5 copies, and the total assay time including 10 min for rapid DNA extraction was approximately 30 min. Data on naturally contaminated raw milk samples indicated that the LAMP method was highly specific and sensitive, giving 100% concordance with Real-time PCR. The results showed that the LAMP-Chemosensor method had the advantages of better sensitivity and speed and less dependence on equipment than the standard (PCR) for specifically detecting low levels of Mycobacterium tuberculosis complex DNA, and this can be useful in the field as a routine diagnostic tool.

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