Inhibition of Non-specific Amplification in Loop-Mediated Isothermal Amplification via Tetramethylammonium Chloride

Abstract

Loop-mediated isothermal amplification (LAMP) may be used in molecular and point-of-care diagnostics for pathogen detection. The amplification occurs under isothermal conditions using up to six primers. However, non-specific amplification is frequently observed in LAMP. Non-specific amplification has the potential to be triggered by forward and reverse internal primers. And the relatively low reaction temperature (55-65 °C) induces the secondary structure via primer-primer interactions. Primer redesign and probe design have been recommended to solve this problem. LAMP primers have strict conditions, such as Tm, GC contents, primer dimer, and distance between primers compared to conventional PCR primers. Probe design requires specialized knowledge to have high specificity for a target. In polymerase chain reaction (PCR), some chemicals or proteins are used for improving specificity and efficiency. Therefore, we hypothesized that additives can suppress the non-specific amplification. In this study, tetramethylammonium chloride (TMAC), formamide, dimethyl sulfoxide, Tween 20, and bovine serum albumin have been used as LAMP additives. In our study, TMAC was presented as a promising additive for suppressing non-specific amplification in LAMP.

Supplementary information: The online version contains supplementary material available at 10.1007/s13206-022-00070-3.

Keywords: Additive; Loop-mediated isothermal amplification; Non-specific amplification; Tetramethylammonium chloride.

Fig. 1

Sensitivity analysis of SARS-CoV-2 primers. 6.8 × 10⁶ copies/µL to 6.8 × 10¹ copies/µL use to analyze LoD. The lowest concentration detected in 20 out of 20 trials was 6.8 × 10² copies/µL. The LoD determined this concentration

Fig. 2

Analysis of ΔCt and Ct_{(TMAC, positive)} according to TMAC concentration. Mean Ct and standard deviation of three tested experiments. A Comparison of ΔCt_(TMAC) and ΔCt_(control). ΔCt_(TMAC) increased with increasing TMAC concentration. A difference between ΔCt_(control) and ΔCt_(TMAC) is more than 30 min. B Ct_{(2.5% TMAC, positive)} and Ct_{(5% TAMC, positive)} did not show a significant difference with Ct_{(control, positive)}. However, Ct_{(7.5% TAMC, positive)} differed from Ct_{(control, positive)} by 4 min

Fig. 3

Analysis of ΔCt and Ct_{(formamide, positive)} according to formamide concentration. Mean Ct and standard deviation of three tested experiments. A Comparison of ΔCt_(formamide) and ΔCt_(control). ΔCt_{(2.5% formamide)} decreased by 0.94 than ΔCt_(control). However, ΔCt_{(5% formamide)} and ΔCt_{(2.5% formamide)} have increased values than ΔCt_(control). B The value of Ct_(positive) increased with increasing formamide concentration. Ct_{(7.5% formamide, positive)} delayed up to 9 min by Ct_{(control, positive)}

Fig. 4

Analysis of ΔCt and Ct_{(DMSO, positive)} according to DMSO concentration. Mean Ct and standard deviation of three tested experiments. A ΔCt_{(2.5% DMSO)} did not show a significant difference with ΔCt_(control). The values of ΔCt_{(5% DMSO)} and ΔCt_{(7.5% DMSO)} increased compared to ΔCt_(control). B All concentrations of Ct_{(DMSO, positive)} were observed increased values than that of Ct_{(control, positive)}. Ct_{(7.5% DMSO, positive)} was delayed to 10 min by Ct_{(control, positive)}

Fig. 5

Analysis of ΔCt and Ct_{(Tween 20, positive)} according to Tween 20 concentration. Mean Ct and standard deviation of three tested experiments. A All ΔCt_{(Tween 20)} except ΔCt_{(0.5% Tween 20)} have decreased values than ΔCt_(control). High concentrations of Tween 20 improve non-specific amplification. B All concentrations of Ct_{(Tween 20, positive)} have a decreased value than Ct_{(control, positive)}. Tween 20 also improves reactivity

Fig. 6

Analysis of ΔCt and Ct_{(BSA, positive)} according to BSA concentration. Mean Ct and standard deviation of three tested experiments. A ΔCt_{(0.1 mg/ml BSA)} and ΔCt_{(0.25 mg/ml BSA)} have increased values than ΔCt_(control). However, high concentrations of BSA decreased the ΔCt value. B The value of Ct_{(BSA, positive)} decreased with increasing BSA concentration