Reham H. Tammam

The effect of gold nanoparticles on the diagnostic polymerase chain reaction technique for equine herpes virus 1 (EHV-1)

Nano-biotechnology has been a noticeable research area because of its successful applications in molecular diagnostics and therapy of various genetic and microbial diseases. Although the polymerase chain reaction (PCR) technique is one of the most highlighted and promising applications in the molecular diagnosis field, it suffers from some drawbacks that affect its efficiency. For instance, as a diagnostic technique for equine herpes virus-1 (EHV-1), conventional PCR could lead to false negative results due to the low viral titer in some samples, which leads to the necessity to improve its sensitivity. In this study, we carried out experiments to determine the effects of 15 nm unmodified citrate-coated gold nanoparticles (GNPs) on the key PCR reactants in order to see if these would enhance the overall outcomes of the reaction. Our results showed that, after optimization of the GNPs, oligonucleotide primers and Taq polymerase concentrations, a specific high yield amplification with a detection limit of 102 DNA copies could be reached compared to the 105 to 104 detection limit of conventional PCR. Thus, the developed and optimized GNPs-assisted PCR technique could be used for a more efficient, highly sensitive molecular detection of EHV-1.

Application of gold nanoparticle-assisted PCR for equine herpesvirus 1 diagnosis in field samples

Equine herpesvirus 1 (EHV-1) is one of the most significant pathogens that affects equine species worldwide, causing sporadic abortion, neonatal deaths, chorioretinopathy, as well as neurological and upper respiratory tract diseases. Currently, conventional PCR targeting different genes is used widely for the molecular detection of EHV-1, but the low viral titer in some clinical samples can lead to false negative results. In this study, we aimed to assess gold nanoparticle (GNP)-assisted PCR as an inexpensive, highly efficient, and sensitive method for the detection of EHV-1, and to compare its results with conventional PCR and real-time quantitative PCR (qPCR). Out of 83 field samples, 28.9%, 26.5%, and 15.6% were EHV-1-positive by qPCR, GNP-assisted PCR and conventional PCR, respectively. All three techniques specifically target the viral glycoprotein B gene. The optimized GNP-assisted PCR showed no cross-reactivity with EHV-1-negative samples (diagnosed by qPCR). GNP-assisted PCR is a powerful new tool for EHV-1 detection and surveillance, because of its simplicity, sensitivity and specificity. It can be used as an alternative to qPCR in laboratories that cannot afford the expense of a qPCR system.