Date Published: December , 2017
Publisher: Makerere Medical School
Author(s): Deguo Wang.
Escherichia coli serogroups O26, O45, O103, O111, O121, O145, and O157 are the causative agents of human diseases, and LAMP assays have been developed for detection of the seven leading STEC serogroups.
The method is based on the principle of the reaction performed by a DNA polymerase with strand displacement activity and a set of two specially designed inner primers (FIP and BIP) and two outer primers (F3 and B3). LAMP is highly specific for the target sequence because six independent sequences (F1c, F2, F3, B1c, B2 and B3) recognize the target sequence in the initial stage and four independent sequences (F1c, F2, B1c, and B2) amplify the target sequence in the later stage of the LAMP reaction. Under an isothermal condition, the amplification efficiency of the LAMP method is extremely high because of the absence of a ramp time for thermal change, because it is an isothermal reaction. Therefore, the LAMP assay has the advantage of specificity, selectivity and rapidity over other nucleic acid amplification methods such as polymerase chain reaction (PCR)2,3, nucleic acid sequence based amplification (NASBA)4,5, strand displacement amplification (SDA)6, rolling circle amplification (RCA)7 as well as helicase dependent amplification (HDA)8. Moreover, Nagamine et al advanced the method by putting forward loop primers (LF and LB) that accelerated the LAMP reaction9.
Real-time PCR instrument is a versatile tool for study on amplification of nucleic acids, it can detemine the melt cure of amplified product for judgement of non-specific amplification, and it had been used to evaluate Wang LAMP assays in the study. Either non-specific amplification or aerosol contamination can result in false positive results of LAMP, but, as our experiment indicated, the melt curve of specific amplification was significantly different with that of non-specific amplification, therefore, the non-specific amplification can be distinguished from that of aerosol contamination, It was found via the amplification plots and melt curves that all Wang LAMP assays had the defect of non-specific amplification.
In summary, we had found that all Wang LAMP assays for detection of 7 main STEC serogroups had the defect of non-specific amplification by aid of Real-time PCR instrument, and we had improved these methods via adding 1% tetramethylene sulfoxide and 5% dimethyl sulfoxide into LAMP reaction mixtures as well as optimizing temperature. These modified LAMP assays can sensitively and specifically detect corresponding main STEC serogroups as commercial Isothermal Amplification Kit does.