Comparison of RNA extraction methods to augment the sensitivity for the differentiation of vesicular stomatitis virus Indiana1 and New Jersey

• Published in: Journal of clinical laboratory analysis Vol. 25; no. 2; pp. 95 - 99
• Main Authors: Arshed, Muhammad Javed, Magnuson, Roberta J., Triantis, Joni, Abubakar, Muhammad, Campen, Hana Van, Salman, Mo
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 2011; John Wiley & Sons, Inc
• Subjects: Animals; Cattle; Cell culture; Differential diagnosis; Differentiation; guanidine-thiocyanate; Humans; Lymph nodes; Lymph Nodes - chemistry; Lymph Nodes - virology; Original; Polymerase chain reaction; Predictive Value of Tests; Purification; rapid detection; Reagent Kits, Diagnostic; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA extraction; RNA, Viral - isolation & purification; RNA-directed DNA polymerase; sensitivity; Vesicular Stomatitis - diagnosis; Vesicular Stomatitis - virology; Vesicular stomatitis Indiana virus - classification; Vesicular stomatitis Indiana virus - genetics; Vesicular stomatitis Indiana virus - isolation & purification; Vesicular stomatitis New Jersey virus - classification; Vesicular stomatitis New Jersey virus - genetics; Vesicular stomatitis New Jersey virus - isolation & purification; Vesicular stomatitis virus; vesicular stomatitis virus and viral diagnosis
• ISSN: 0887-8013; 1098-2825; 1098-2825
• DOI: 10.1002/jcla.20439

Two methods for the extraction of RNA of vesicular stomatitis virus (VSV) Indiana1 and New Jersey and their simultaneous amplification by one‐step polymerase chain reaction using reverse transcriptase were evaluated. A guanidine‐thiocyanate‐based RNA extraction (Qiagen RNeasy Mini Kit, Qiagen, Valencia, CA ) followed by column‐based purification coupled with one‐step RT‐PCR proved to be a simple, safe, practicable, and reliable tool for rapid, highly sensitive, and specific differential diagnosis of both types of VSV in cell lysate and spiked tissue samples as compared with the tri‐phasic extraction method (Tri‐reagent method). When RNA was extracted either from VSV cell culture stock or from VSV spiked bovine lymph nodes by using Qiagen RNeasy Mini Kit, the detection limit in the multiplex RT‐PCR was as low as 0.505 to 2.84 TCID50 for VSV‐IND and VSV‐NJ, respectively. The multiplex RT‐PCR consistently detected VSV‐IND and NJ RNA in as little as 0.1–1.0 fg of total RNA from spiked BHK‐21 cell suspension when Qiagen RNeasy mini kit was used. The multiplex RT‐PCR assay was capable of detecting both types of VSV in a one‐step reaction tube. The minimum sensitivity of this assay in various experiments was 0.1683 TCID50 (IND), 0.0946 TCID50 (NJ), and 0.057 fg (IND and NJ) per 2 µl PCR sample, which is significantly more sensitive than reported previously (0.28–2.8 TCID50/1 µl). So the present study improved the sensitivity of previously reported multiplex RT‐PCR for the detection and differentiation of VSV‐IND and VSV‐NJ in a single assay. J. Clin. Lab. Anal. 25:95–99, 2011. © 2011 Wiley‐Liss, Inc.