Preliminary Evaluation of Rapid Visual Identification of Burkholderia pseudomallei Using a Newly Developed Lateral Flow Strip-Based Recombinase Polymerase Amplification (LF-RPA) System

Abstract

Burkholderia pseudomallei is an important infectious disease pathogen that can cause melioidosis. Melioidosis is mainly prevalent in Thailand, northern Australia and southern China and has become a global public health problem. Early identification of B. pseudomallei is of great significance for the diagnosis and prognosis of melioidosis. In this study, a simple and visual device combined with lateral flow strip-based recombinase polymerase amplification (LF-RPA) was developed, and the utility of the LF-RPA assay for identifying B. pseudomallei was evaluated. In order to screen out the optimal primer probe, a total of 16 pairs of specific primers targeting the orf2 gene of B. pseudomallei type III secretion system (T3SS) cluster genes were designed for screening, and F1/R3 was selected as an optimal set of primers for the identification of B. pseudomallei, and parameters for LF-RPA were optimized. The LF-RPA can be amplified at 30-45°C and complete the entire reaction in 5-30 min. This reaction does not cross-amplify the DNA of other non-B. pseudomallei species. The limit of detection (LOD) of this assay for B. pseudomallei genomic DNA was as low as 30 femtograms (fg), which was comparable to the results of real-time PCR. Moreover, 21 clinical B. pseudomallei isolates identified by 16S rRNA gene sequencing were retrospectively confirmed by the newly developed LF-RPA system. Our results showed that the newly developed LF-RPA system has a simple and short time of operation and has good application prospect in the identification of B. pseudomallei.

Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a

“…The specificity of the crBP34-DETECTR may arise from the designed crRNA—crBP34, which was a result of large-scale phylogenetics and bioinformatics analyses to remove cross-reactivity ( Fig 1B and 1C ). This contrasts with previously reported detection assays using real-time PCR [ 21 – 25 ], LAMP [ 26 , 27 ] or RPA [ 28 – 30 ], in which primers were designed based on selected genomic loci or limited bioinformatics analysis on few reference strains. Testing crBP34 and the other crRNAs with B .…”

“…Although these methods showed high sensitivity and specificity, real-time PCR-based assays require expensive real-time PCR machines, which may not be readily available in many laboratories. As alternatives, isothermal DNA amplifications such as loop-mediated isothermal amplification (LAMP) [26,27] and recombinase polymerase amplification (RPA) [28][29][30] have been developed to overcome this dependency on expensive equipment. Both assays exhibit very sensitive detection.…”

Specific Detection and Enumeration of Burkholderia cepacia Complex by Flow Cytometry Using a Fluorescence-Labeled Oligonucleotide Probe

“…These tests are mainly limited by their low sensitivity and have been shown to be relatively inaccurate with regards to the detection of BCC [4,13]. To overcome this sensitivity challenge, polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and droplet digital PCR (ddPCR) have also been utilized to test for the presence of BCC [13][14][15][16][17][18][19][20]. PCR has been shown to be rapid, simple, easy to use, and relatively inexpensive for the detection of BCC [4].…”

“…PCR has been shown to be rapid, simple, easy to use, and relatively inexpensive for the detection of BCC [4]. LAMP and RPA are efficient isothermal methods that eliminate the need for a thermal cycler and have high deployment potential in resource-limited settings [4,[18][19][20]. Two major limitations of qPCR are of particular concern, namely its inability to distinguish live from dead cells and the amplification reactions sensitivity to inhibitors.…”

Development and evaluation of a rapid and sensitive multienzyme isothermal rapid amplification with a lateral flow dipstick assay for detection of Acinetobacter baumannii in spiked blood specimens

“…They can amplify complex DNA targets at room temperature within 10 min, which is more advantageous than other isothermal amplification methods ( Shang et al., 2021 ; Sun et al., 2021 ; Tu et al., 2022 ). In the detection format of MIRA amplifier, lateral flow dipstick (LFD) based MIRA detection has been widely established for various detection target ( Li et al., 2019a ; Li et al., 2021 ). In order to meet the needs of rapid detection of first aid and emergency treatment, especially for laboratories with limited resources and poor equipment, MIRA-LFD assay is an ideal choice.…”