Diverse methods of reducing and confirming false-positive results of loop-mediated isothermal amplification assays: A review

Kim, So-Hee; Lee, So‐Young; Kim, Unji; Oh, Se‐Wook

doi:10.1016/j.aca.2023.341693

Cited by 27 publications

(9 citation statements)

References 67 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Nontemplate amplification (nonspecific amplification and fluorescence in the absence of any template) has been reported in LAMP assays sporadically. It is thought to be more common in LAMP primers due to the additional bases and length compared with PCR primers [ 28 , 29 ]. In earlier research [ 10 ] comparing this ASFV LAMP with qPCR, there was also a small number of LAMP results with nonspecific fluorescence in sample wells, where matching qPCR results were consistently negative.…”

Section: Resultsmentioning

confidence: 99%

Stability of Genotube® Swabs for African Swine Fever Virus Detection Using Loop-Mediated Isothermal (LAMP) Laboratory Testing on Samples Stored without Refrigeration

Phillips,

Conceicao,

Jong

et al. 2024

Viruses

View full text Add to dashboard Cite

African swine fever (ASF) is a transboundary viral disease which causes high mortality in pigs. In many low- and middle-income countries and in remote areas where diagnostic surveillance for ASF virus (ASFV) is undertaken, access to trained animal health technicians, sample collection, cold chain storage and transport of samples to suitably equipped laboratories can be limiting when traditional sampling and laboratory tests are used. Previously published studies have demonstrated that alternative sampling matrices such as swabs and filter papers can be tested using PCR without refrigeration for up to a week. This study used Genotube® swabs stored in temperate and tropical climates without refrigeration for four weeks after collection to demonstrate there was no change in test performance and results using loop-mediated isothermal amplification (LAMP) ASFV detection on a series of pig serum samples including serum spiked with a synthetic ASFV positive control, naturally acquired ASFV positive serum from Timor-Leste and negative ASFV serum samples. The use of Genotube® swabs for ASFV detection for surveillance purposes, coupled with testing platforms such as LAMP, can provide an alternative to traditional testing methodology where resources are limited and time from collection to testing of samples is prolonged.

show abstract

Section: Resultsmentioning

confidence: 99%

Stability of Genotube® Swabs for African Swine Fever Virus Detection Using Loop-Mediated Isothermal (LAMP) Laboratory Testing on Samples Stored without Refrigeration

Phillips,

Conceicao,

Jong

et al. 2024

Viruses

View full text Add to dashboard Cite

show abstract

“…Due to the relative novelty of LAMP, several studies have been published attempting to improve its performance, and they were mainly focused on reducing unspecific amplifications by supplementing the reactions with different types of chemicals [ 47 ]. Moving forward in the comparison, it was decided to evaluate the effect of five typical LAMP supplements on each one of the mixes.…”

Section: Discussionmentioning

confidence: 99%

“…It is worth mentioning that in the comparison against the native reaction, the supplement that least impacted the Tt was pullulan, in agreement with what was previously reported by Gao et al [ 29 ], who also demonstrated its compatibility with other isothermal techniques such as CPA and RCA [ 49 ]. None of the supplements accelerated NEB for any of the targets, while betaine and DMSO, particularly the latter, did reduce the amplification time of Lava and Syn when targeting ttr , but not the other two targets, which may be related to a decrease in the melting as this target was determined to have a higher %GC; see Table 3 [ 28 , 47 , 50 , 51 , 52 ]. Jena was enhanced by the addition of DMSO when targeting hly , and OG with pullulan, which also enhanced the amplification speed targeting rfbE with this mix even though less than TMAC and GuHCl as shown in Figure 3 b,d,f.…”

Section: Discussionmentioning

confidence: 99%

Evaluating Commercial Loop-Mediated Isothermal Amplification Master Mixes for Enhanced Detection of Foodborne Pathogens

Costa-Ribeiro,

Lamas,

Garrido-Maestu

2024

Foods

View full text Add to dashboard Cite

Loop-mediated isothermal amplification, LAMP, is nowadays the most popular isothermal nucleic acid amplification technique, and as such, several commercial, ready-to-use master mixes have flourished. Unfortunately, independent studies to determine their performance are limited. The current study performed an independent evaluation of the existing ready-to-use commercial LAMP master mixes WarmStart® LAMP Kit, LavaLAMP™ DNA Master Mix, Saphir Bst Turbo GreenMaster, OptiGene Fast Master Mix ISO-004, and SynLAMP Mix. To reduce bias, three different genes, namely ttr (Salmonella spp.), rfbE (E. coli O157), and hly (Listeria monocytogenes), were targeted. The comparison was based on amplification speed, performance with decreasing DNA concentrations, and the effect of five typical LAMP reaction additives (betaine, DMSO, pullulan, TMAC, and GuHCl). Significant differences were observed among the different master mixes. OptiGene provided the fastest amplification and showed less detrimental effects associated with the supplements evaluated. Out of the chemicals tested, pullulan provided the best results in terms of amplification speed. It is noteworthy that the different additives impacted the master mixes differently. Overall, the current study provides insights into the performance of commercial LAMP master mixes, which can be of value for the scientific community to better select appropriate reagents when developing new methods.

show abstract

“…In this manner, aerosol pollution of the RPA amplicons was inevitable, 31,32 which may cause false positives. 33,34 Besides, the postamplification addition of reagents also complicates the overall storage and transportation and thus is difficult to be deployed to the field.…”

Section: ■ Introductionmentioning

confidence: 99%

“…However, the chromogenic reaction of TMB needs to be carried out in weakly acidic media (pH = 4.5), thus cap opening was required for RPA amplicons to be quantified. In this manner, aerosol pollution of the RPA amplicons was inevitable, , which may cause false positives. , Besides, the postamplification addition of reagents also complicates the overall storage and transportation and thus is difficult to be deployed to the field.…”

Section: Introductionmentioning

confidence: 99%

Integrating Recombinase Polymerase Amplification and Photosensitization Colorimetric Detection in One Tube for Fast Screening of C. sakazakii in Formula Milk Powder

Wang,

Zheng,

et al. 2024

Anal. Chem.

View full text Add to dashboard Cite

Cronobacter sakazakii (C. sakazakii) is a widely existing opportunistic pathogen and thus threatens people with low immunity, especially infants. To prevent the outbreak, a rapid and accurate on-site testing method is required. The current standard culture-based method is time-consuming (3−4 days), while the nucleic acid amplification (PCR)based detection is mostly carried out in central laboratories. Herein, isothermal recombinase polymerase amplification (RPA) coupled with a photosensitization colorimetric assay (PCA) was adopted for the on-site detection of C. sakazakii in powdered infant formulas (PIFs). The lowest visual detection concentration of C. sakazakii is 800 cfu/mL and 2 cfu/g after 8 h bacteria pre-enrichment. Furthermore, to avoid typical cap opening-resulted aerosol pollution, the PCA reagents were lyophilized onto the cap of the RPA tube (containing lyophilized RPA reagents). After amplification, the tube was subjected to simple shaking to mix the PCA reagents with the amplification products for lightdriven color development. Such a one-tube assay offered a lowest concentration of 1000 copies of genomic DNA of C. sakazakii within 1 h. After 8 h of bacterial enrichment, the lowest detecting concentration could be pushed down to 5 cfu/g bacteria in PIF. To facilitate on-site monitoring, a portable, battery-powered PCA device was designed to mount the typical RPA 8-tube strip, and a color analysis cellphone APP was further employed for facile readout.

show abstract

Diverse methods of reducing and confirming false-positive results of loop-mediated isothermal amplification assays: A review

Cited by 27 publications

References 67 publications

Stability of Genotube® Swabs for African Swine Fever Virus Detection Using Loop-Mediated Isothermal (LAMP) Laboratory Testing on Samples Stored without Refrigeration

Stability of Genotube® Swabs for African Swine Fever Virus Detection Using Loop-Mediated Isothermal (LAMP) Laboratory Testing on Samples Stored without Refrigeration

Evaluating Commercial Loop-Mediated Isothermal Amplification Master Mixes for Enhanced Detection of Foodborne Pathogens

Integrating Recombinase Polymerase Amplification and Photosensitization Colorimetric Detection in One Tube for Fast Screening of C. sakazakii in Formula Milk Powder

Contact Info

Product

Resources

About