Effect of primer binding sites mutations on the efficacy of measles virus RNA detection using PCR and LAMP techniques

INTRODUCTION. The high variability of the measles virus RNA genome poses risks of mutations accumulating at primer binding sites (complementary binding site), potentially impacting the reliability of molecular measles diagnostics. In this study, we have assessed the impact of such mutations on the efficiency of two molecular genetic methods: PCR and loop-mediated isothermal amplification (LAMP).

AIM. This study aimed to evaluate the occurrence of mutations at primer binding sites and their impact on the efficiency of PCR and LAMP methods in detecting measles virus RNA.

MATERIALS AND METHODS. Clinical samples of nasopharyngeal secretions (LabQuest, Moscow, Russia) and nucleotide sequences from NCBI database were used in the study. AmpliTest RIBO-prep kit was used for RNA extraction. AmpliTest Measles kit and LAMP-based kit were used for amplification. MAFFT, Jalview 2.1, CD-HIT, MEGA1, and FigTree v.1.4.3 software were used to perform a bioinformatics analysis.

RESULTS. Having analyzed 1,080 nucleotide sequences of the measles virus, we found no more than one mutation in the target primers target sites and probe used for PCR. The analyzed LAMP primer binding sites showed greater variability compared to PCR primers; even in this case, 96.5% of the sequences contained no more than a single mutation at each site. Out of 69 clinical samples, only 15 had one mutation in the forward primer binding site, with a minor impact on PCR performance at low virus concentrations. For the LAMP method, three mutations were detected in the F3, B1c, and B2 primer target sites in 18 samples. In a model experiment, the mismatch in the region of the outer primer F3 did not affect the speed and sensitivity of the reaction. Inversely, the loop forming primer BIP performed abnormally well in the presence of two mutations in the RNA sequences. This can indirectly prove the existence of conformational aspects for long loop primers.

CONCLUSIONS. Both methods (PCR and LAMP) effectively detect samples with mutations; however, the LAMP method warrants further research to expand its potential for diagnosing measles.

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