Establishment of the molecular beacon-loop-mediated isothermal amplification method for the rapid detection of Porphyromonas gingivalis

Yang

et al. 2020

Sci Rep

The cytochrome cd1-containing nitrite reductase, nirS, plays an important role in biological denitrification. Consequently, investigating the presence and abundance of nirS is a commonly used approach to understand the distribution and potential activity of denitrifying bacteria, in addition to denitrifier communities. Herein, a rapid method for detecting nirS gene with loop-mediated isothermal amplification (LAMP) was developed, using Pseudomonas aeruginosa PAO1 (P. aeruginosa PAO1) as model microorganism to optimize the assay. The LAMP assay relied on a set of four primers that were designed to recognize six target sequence sites, resulting in high target specificity. The limit of detection for the LAMP assay under optimized conditions was 1.87 pg/reaction of genomic DNA, which was an order of magnitude lower than that required by conventional PCR assays. Moreover, it was validated that P. aeruginosa PAO1 cells as well as genomic DNA could be directly used as template. Only 1 h was needed from the addition of bacterial cells to the reaction to the verification of amplification success. The nirS gene of P. aeruginosa PAO1 in spiked seawater samples could be detected with both DNA-template based LAMP assay and cell-template based LAMP assay, demonstrating the practicality of in-field use.

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Rapid detection of cytochrome cd1-containing nitrite reductase encoding gene nirS of denitrifying bacteria with loop-mediated isothermal amplification assay

Yang

et al. 2020

Sci Rep

“…One important development of LAMP techniques has been to quantitatively assess targets (qLAMP) [20]. These developments have led to stable linear determinations of products across as many as nine orders of magnitude concentration over a wide range of human and agricultural pathogens [20–23]. Such quantitative assessments could be useful for Wolbachia monitoring, because a key feature of release success is the frequency of the endosymbiont in field populations.…”

Section: Introductionmentioning

confidence: 99%

A LAMP assay for the rapid and robust assessment of Wolbachia infection in Aedes aegypti under field and laboratory conditions

et al. 2019

With Wolbachia-based arbovirus control programs being scaled and operationalised around the world, cost effective and reliable detection of Wolbachia in field samples and laboratory stocks is essential for quality control. Here we validate a modified loop-mediated isothermal amplification (LAMP) assay for routine scoring of Wolbachia in mosquitoes from laboratory cultures and the field, applicable to any setting. We show that this assay is a rapid and robust method for highly sensitive and specific detection of wAlbB Wolbachia infection within Aedes aegypti under a variety of conditions. We test the quantitative nature of the assay by evaluating pooled mixtures of Wolbachia-infected and uninfected mosquitoes and show that it is capable of estimating infection frequencies, potentially circumventing the need to perform large-scale individual analysis for wAlbB infection status in the course of field monitoring. These results indicate that LAMP assays are useful for routine screening particularly under field conditions away from laboratory facilities.

“…However, these dyes lack specificity, and can hardly provide direct evidence of specific amplification, especially in multiplex LAMP which amplifies multiple targets simultaneously. To extend the current dLAMP to multiplex detections, we suggest using specific probes, such as molecular beacon, to identify the specific amplification product …”

Section: Numerical and Statistical Analysis In The Dlamp Techniquementioning

confidence: 99%

Droplet and Microchamber‐Based Digital Loop‐Mediated Isothermal Amplification (dLAMP)

Yuan

Chao

Shum

2020

Small

Digital loop‐mediated isothermal amplification (dLAMP) refers to compartmentalizing nucleic acids and LAMP reagents into a large number of individual partitions, such as microchambers and droplets. This compartmentalization enables dLAMP to be an excellent platform to quantify the absolute number of the target nucleic acids. Owing to its low requirement for instrumentation complexity, high specificity, and strong tolerance to inhibitors in the nucleic acid samples, dLAMP has been recognized as a simple and accurate technique to quantify pathogenic nucleic acid. Herein, the general process of dLAMP techniques is summarized, the current dLAMP techniques are categorized, and a comprehensive discussion on different types of dLAMP techniques is presented. Also, the challenges of the current dLAMP are illustrated together with the possible strategies to address these challenges. In the end, the future directions of the dLAMP developments, including multitarget detection, multisample detection, and processing nucleic acid extraction are outlined. With recently significant advances in dLAMP, this technology has the potential to see more widespread use beyond the laboratory in the future.