An integrated direct loop-mediated isothermal amplification microdevice incorporated with an immunochromatographic strip for bacteria detection in human whole blood and milk without a sample preparation step

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: Sensitivity Of Cell-template Based Lamp Assaymentioning

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

“…Through miniaturization and integration of various bioanalytical processes, microfluidic technologies have the potential to offer rapid, cost-effective, and portable solutions to medical needs in the developing world [10]. Within the application space of enteric screening, a variety of microfluidic detection modalities have been reported, including electrochemical [11,12], surface plasmon resonance (SPR) [13], and molecular methods such as polymerase chain reaction (PCR) [14,15,16] and loop-mediated isothermal amplification (LAMP) [17,18,19,20,21,22,23]. A notable subset of the microfluidic system types is centrifugal, or lab-on-a-disc, devices, first developed in the late 1990s [24].…”

Section: Introductionmentioning

confidence: 99%

Rapid, Portable, Multiplexed Detection of Bacterial Pathogens Directly from Clinical Sample Matrices

et al. 2016

Enteric and diarrheal diseases are a major cause of childhood illness and death in countries with developing economies. Each year, more than half of a million children under the age of five die from these diseases. We have developed a portable, microfluidic platform capable of simultaneous, multiplexed detection of several of the bacterial pathogens that cause these diseases. This platform can perform fast, sensitive immunoassays directly from relevant, complex clinical matrices such as stool without extensive sample cleanup or preparation. Using only 1 µL of sample per assay, we demonstrate simultaneous multiplexed detection of four bacterial pathogens implicated in diarrheal and enteric diseases in less than 20 min.

“…LAMP-based methods show great promise for use in POC devices and improvements to enhance the ease of use and assay robustness. Lee et al [ 153 ] pioneered a direct LAMP technique to eliminate the genomic extraction process by utilizing a PCR buffer capable of lysing bacterial cell membrane and inactivating PCR inhibitors in human whole blood or milk. They demonstrated single-cell detection of Staphylococcus aureus and E. coli in human whole blood or milk within a 1-h processing time.…”

Section: Amplificationmentioning

confidence: 99%

Towards Multiplex Molecular Diagnosis—A Review of Microfluidic Genomics Technologies

Basha

Yousuff

et al. 2017

Micromachines

Highly sensitive and specific pathogen diagnosis is essential for correct and timely treatment of infectious diseases, especially virulent strains, in people. Point-of-care pathogen diagnosis can be a tremendous help in managing disease outbreaks as well as in routine healthcare settings. Infectious pathogens can be identified with high specificity using molecular methods. A plethora of microfluidic innovations in recent years have now made it increasingly feasible to develop portable, robust, accurate, and sensitive genomic diagnostic devices for deployment at the point of care. However, improving processing time, multiplexed detection, sensitivity and limit of detection, specificity, and ease of deployment in resource-limited settings are ongoing challenges. This review outlines recent techniques in microfluidic genomic diagnosis and devices with a focus on integrating them into a lab on a chip that will lead towards the development of multiplexed point-of-care devices of high sensitivity and specificity.