Detection of nucleic acids and elimination of carryover contamination by using loop-mediated isothermal amplification and antarctic thermal sensitive uracil-DNA-glycosylase in a lateral flow biosensor: application to the detection of Streptococcus pneumoniae

Abstract

The authors report on a loop-mediated isothermal amplification (LAMP) scheme that uses antarctic thermally sensitive uracil-DNA-glycosylase (AUDG) for simultaneous detection of nucleic acids and elimination of carryover contamination. It was applied in a lateral flow assay (LFA) format. The assay has attractive features in that it does not require the use of labeled primers or probes, and can eliminate false-positive results generated by unwanted hybridization between two labeled primers or between a labeled primer and probe. LAMP amplification and AUDG digestion are conducted in a single pot, and the application of a closed-tube reaction prevents false-positives due to carryover contamination. The method was applied to the detection of the human pathogen Streptococcus pneumoniaein in pure cultures and spiked blood samples. This LFA can detect S. pneumoniae in pure cultures with a 25 fg.μL detection limit and in spiked blood samples with a 470 cfu.mL detection limit. Conceivably, this assay can be applied to the detection of various other targets if the specific LAMP primers are available. Graphical abstract ᅟ.

Validation of a Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Invasive Pneumococcal Disease

“…Minor differences between LAMP sensitivities could be explained by the higher number of replicates that we used for LOD calculation, as suggested by guidelines (FDA, 2001), in comparison to the number of replicates considered in the referenced studies (<3). The LOD described in our study is also in concordance with the value of 100-500 CFU/mL (between 10 3 and 10 4 cp/mL) targeting the 16S rRNA gene (Huy et al, 2012) and the ply gene (Wang et al, 2018). Conversely, another published work established a LOD about 300 pg/µL (10 5 copies/µl) for a specific LAMP test based on SYBR Green fluorophore and using a portable tube scanner (Xia et al, 2014).…”

“…It should be noted that total time required for PCR amplification could be reduced with cycle alterations slightly below 1 h. However, we followed the PCR protocol recommended by the CDC to ensure adequate sensitivity and specificity while achieving much faster time of amplification with LAMP. The LAMP reaction is a single tube technique for DNA amplification that does not require thermal cyclers (Huy et al, 2012;Xia et al, 2014;Wang et al, 2018). In addition, amplification can be detected by turbidimetry or visual color change without the need for any other equipment (Huy et al, 2012).…”

Detection of Streptococcus Pneumoniae and elimination of carryover contamination by multiple cross displacement amplification coupled with antarctic thermal sensitive uracil-DNA-glycosylase

“…pneumoniae as low as 447 CFU/ml (equivalent to 4.47 CFU per reaction), which is similar to that of LAMP [19]. Thus, ply-MCDA assay was a sensitive method for S. pneumoniae detection.…”

“…The detection limit of ply-MCDA assay was 10fg (Fig.3). It has been reported that LAMP can detect S. pneumoniae as few as 25 fg [19] or 20 fg [20]. We also assessed the clinical sensitivity of this assay by using spiked sputum samples.…”

“…The results of this study suggested that trace amounts of carryover contamination (1×10−19 g/μL) can lead to false positive results. Thus, AUDG enzyme digestion was performed to prevent the false positive results caused by carryover contamination, which specifically cleave uracil bases and permit nature templates to be amplified normally [11,19], and which can eliminate up to 1×10−15 g/μL of simulated contaminants. These results suggested that ply-MCDA-AUDG assay can effectively eliminate carryover contamination and then reduce false positive results.…”