Comparison of multiplex RT‑PCR and real‑time HybProbe assay for serotyping of dengue virus using reference strains and clinical samples from India.

Background: Dengue virus serotyping is crucial from clinical management and epidemiological point of view. Aims: To compare efficacy of two molecular detection and typing methods, namely, multiplex reverse transcription polymerase chain reaction (RT‑PCR) and real‑time Hybprobe assay using a panel of known dilution of four reference Dengue virus strains and a panel of sera collected from clinically suspected dengue patients. Settings: This study was conducted at a tertiary‑care teaching hospital in Delhi, India. Materials and Methods: Dengue serotype specific virus strains were used as prototypes for serotyping assays. Viral load was quantified by quantitative real time reverse transcription polymerase chain reaction (qRT‑PCR). Acute phase serum samples were collected from 79 patients with clinically suspected Dengue fever on their first day of presentation during September‑October 2012. Viral RNA from serum and cell culture supernatant was extracted. Reverse transcription was carried out. Quantitative detection of DENV RNA from reference strain culture supernatants and each of the 79 patient samples by real‑time PCR was performed using light cycler Taqman master mix kit. Serotyping was done by multiplex RT‑PCR assay and Hybprobe assay. Results: The multiplex RT‑PCR assay, though found to be 100% specific, couldn’t serotype either patient or reference strains with viral load less than 1000 RNA copies/ml. The Hybprobe assay was found to have 100% specificity and had a lower limit of serotype detection of merely 3.54 RNA copies/ml. Conclusions: HybProbe assay has an important role especially in situations where serotyping is to be performed in clinical samples with low viral load.

Identification of Brucella melitensis isolates originating from Mongolia and diagnostic real-time PCR evaluation using a specific SNP / 대한수의학회지

A real-time PCR assay using hybridization probe (HybProbe) has been developed to detect Brucella (B.) melitensis strains. The primer and HybProbe sets were designed based on the gap gene of chromosome I with a specific single nucleotide polymorphism of B. melitensis. Specificity of the assay was confirmed by comparison to reference Brucella species and other related strains. In the melting curve analysis, B. melitensis generated a peak at 67degrees C unlike those for other Brucella species observed at 61degrees C. Sensitivity of the assay for B. melitensis ranged from 20 ng to 200 fg of genomic DNA. The ability to identify 94 Mongolian B. melitensis isolates using the real-time PCR assay was identical to that of classical biotyping methods and differential multiplex PCR. These data showed that this new molecular technique is a simple and quick method for detecting B. melitensis, which will be important for the control and prevention of brucellosis.