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

Abstract

PurposeThis study aimed to establish the multienzyme isothermal rapid amplification with a lateral flow dipstick (MIRA-LFD) assay and evaluate its performance in detection of A. baumannii in spiked blood specimens.MethodsThe study was divided into two stages: a pilot study to establish the methodology and a clinical validation study to evaluate its performance. In the first step, we designed primers specific to detect A. baumannii, optimized the MIRA-LFD assay and analyzed its performance regarding limits of detection, reproducibility, specificity, and efficiency of detection using real-time PCR method. In the second step, we obtained 50 spiked blood isolates and detected these pathogens by MIRA-LFD assay. The MIRA-LFD time was 15 min from DNA sample amplification to complete pathogen detection.ResultsThe developed MIRA-LFD assay displayed a detection limit of 6 CFU/mL for detecting A. baumannii, which was significantly better than that of real-time PCR method, and no cross-reactivity was observed in other non-A. baumannii studied. The results obtained with 50 spiked blood isolates suggested that the developed MIRA-LFD assay had high specificity and sensitivity for identifying A. baumannii.ConclusionsThis study demonstrates that the established MIRA-LFD assay is time-saving, more effective and sensitive, which may become a powerful tool for rapid and reliable diagnosis of bloodstream infection caused by A. baumannii in primary hospitals.

Rapid and Sensitive Detection of Streptococcus iniae in Trachinotus ovatus Based on Multienzyme Isothermal Rapid Amplification

“…The amplification process from both MIRA takes about 20 min or less. Again, they showed well sensitivity as real-time PCR, ten times more than PCR, and rapidity and convenient, less equipment requirement, especially for the grass-roots level such as aquaculture farms with limited resources [23][24][25][26][27].…”

“…In order to detect pathogenic bacteria, many detection methods have been established. PCR is undoubtedly widely used in the detection of pathogenic bacteria, including Staphylococcus aureus [29], salmonella [30], Vibrio cholerae [31], Acinetobacter baumannii [24], Phytophthora sojae [32], and Pseudomonas aeruginosa [33]. With the continuous development of molecular diagnosis, many thermostatic amplification techniques such as LAMP, NASBA [34], and HDA [35] are gradually applied to the diagnosis of pathogenic bacteria.…”

Recent Uses of Paper Microfluidics in Isothermal Nucleic Acid Amplification Tests

“…It is challenging to generalize the reaction time since each assay is designed differently. However, MIRA appears to have the fastest average total assay time of 18.75 min [ 68 , 70 , 72 , 74 ]. One review paper summarized the same characteristics of various isothermal amplification methods.…”

“…The use of helicase (gp41) and recombinases ( Streptomyces coelicolor recA, SC-recA) allows for rapid amplification that can happen between 10–30 min at a constant temperature between 35–42 °C [ 68 , 69 ]. MIRA has also been demonstrated with paper strips and paper microfluidic platforms [ 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 ]. Like the RAA, all paper-based MIRA assays utilized LFIA with colorimetric detection.…”

Rapid visualization molecular fluorescence detection of methicillin‐resistant Staphylococcus aureus using the multiplex MIRA‐qPCR method

“…It is a thermostatic, rapid, nucleic acid–amplification technique that relies on the synergistic action of multiple functional proteins to achieve rapid nucleic acid amplification at room temperature. There have been several studies on the detection of specific organisms (e.g., coronavirus, [ 21 ] hepatitis B, [ 22 ] and Acinetobacter baumannii [ 23 ] ) based on MIRA technology in combination with lateral flow dipsticks recently. But the problems are that the test strips can be easily contaminated resulting in false‐negative results, and aerosol contamination from opening the cap.…”