Construction of Newcastle disease virus LaSota strain-based internal sample for rabies diagnosis with RT-PCR

Introduction. The following factors can impact the reliability of polymerase chain reaction diagnosis: operator errors, amplifier malfunction, presence of reaction inhibitors in the sample, poor reagent quality and others. All this can lead to the so-called false negative results.

Objective. Construction of the internal control based on heterologous Newcastle disease virus for detection of rabies virus by polymerase chain reaction.

Materials and methods. Dry live vaccine against Newcastle disease based on LaSota strain produced by the Federal Centre for Animal Health (Russia) was used as an internal control. RNA extraction from samples was performed with “Ribosorb” reagent kit (Central Research Institute for Epidemiology of the Rospotrebnadzor, Russia). Promega Corporation reagents (USA) and oligonucleotides manufactured by the Syntol Company (Russia) were used for the reverse transcription polymerase chain reaction.

Results. LaSota strain of Newcastle disease virus was selected as the target for the internal control. The primers were designed. Experiments showed that the PCR system for the internal control did not compete with the PCR system for the rabies virus when they were used together.The main parameters of reverse transcription and polymerase reaction were optimized. The developed method was validated using several key parameters: correctness, specificity, sensitivity, repeatability (intermediate precision under same conditions), and reproducibility (intermediate precision under different conditions). Validation results have shown that the method characteristics comply with the required ones.

Conclusion. Newcastle disease virus LaSota strain-based internal control has been constructed for use together with reverse transcription polymerase chain reaction assay for rabies virus detection that allows control of the assay stages in each reaction tube. This internal control after its proper optimization can be also used in experimental studies carried out at relevant research institutions for PCR diagnosis of the diseases caused by other RNA-containing viruses.

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