A Practical Tissue Sampling Method Using Ordinary Paper for Molecular Detection of Infectious Bursal Disease Virus RNA by RT-PCR

Abstract

A practical sampling method for bursal tissue using ordinary paper for molecular diagnosis of infectious bursal disease (IBD) was established. IBD virus-infected bursa was directly smeared on chromatography paper, filter paper, or stationery copy paper and was then fixed with absolute ethanol, Tris-HCl-saturated phenol, or phenol:chloroform:isoamyl alcohol (25:24:1). Flinders Technology Associates (FTA) card, which is designed for the collection of biological samples for molecular detection, was also used. After storage at 37 C for up to 30 days, total RNA directly extracted from the tissue fixed on the papers and FTA card were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of IBD virus (IBDV) RNA. In addition, the ability of each chemical used in the fixation and the FTA card to inactivate IBDV was evaluated. Regardless of the paper quality, storage period, and fixation method, IBDV RNA was consistently detected in all of the samples. IBDV in the bursal tissue was inactivated with phenol but not with ethanol or the unknown chemicals in FTA card. These results show that ordinary papers sustain the viral RNA, as does FTA card, but phenol fixation is superior to FTA card in inactivating IBDV. The new sampling method using ordinary paper with phenol fixation is safe, inexpensive, simple, and easy, and is thus suitable for conducting a global survey of IBD even where laboratory resources are limited. This practical method should contribute to the control of IBD worldwide.

A systematic review of FTA cards® as a tool for viral RNA preservation in fieldwork: Are they safe and effective?

“…The targeted viral genomes belonged to all classes of RNA virus of the Baltimore Classification except Class VI, with 8.5% of the studies reporting amplification of RNA fragments of Class III virus (dsRNA), 68.1% of Class IV virus ((+) ssRNA), 21.3% of Class V virus ((−) ssRNA), and 2.1% of the three classes mentioned above. An animal specimen source accounted for 38.3% of the reports (Moscoso et al, 2005;Maw et al, 2006;Moscoso et al, 2006;Perozo et al, 2006;Purvis et al, 2006;Inoue et al, 2007;Narayanan et al, 2010;Abdelwhab et al, 2011;Kraus et al, 2011;Keeler et al, 2012;Linhares et al, 2012;Madhanmohan et al, 2013;Awad et al, 2014;Biswal et al, 2016;Foss et al, 2016;Jozwiak et al, 2016;Madhanmohan et al, 2016;Manswr et al, 2018). Among them, twelve reports isolated RNA from viruses that can cause disease in poultry or wild birds, like Newcastle virus disease, avian influenza, or infectious bursal disease (Table 1).…”

“…Infectious virus was isolated from one of only fourteen included studies that tested for infectivity of the virus from an FTA card® (Maw et al, 2006). In this study, ADVANTEC chromatography paper No.…”

“…Briefly, viral RNA was obtained by soaking fragments of the FTA card® in an eluent solution, followed by incubation with or without agitation with temperatures that ranged between 4-95°C. After elution, the RNA underwent a purification step using commercial kits (Moscoso et al, 2005;Ndunguru et al, 2005;Maw et al, 2006;Moscoso et al, 2006;Purvis et al, 2006;Abdelwhab et al, 2011;Keeler et al, 2012;Li et al, 2012;Bankamp et al, 2013;Sakai et al, 2015;Tam et al, 2015;Yang et al, 2015;Biswal et al, 2016;Foss et al, 2016;Madhanmohan et al, 2016;Karavina and Gubba, 2017;Manswr et al, 2018) or standard protocols like sodium acetate and isopropanol precipitation (Grund et al, 2010;Price et al, 2014). In one case, magnetic beads were used for purification and concentration of RNA (Linhares et al, 2012) Direct virus RNA purification was performed in 30.6% of the studies.…”

“…Nevertheless, the possibility remains that some infectious virus could be present in this type of sample, as shown by the study of bursal disease virus. In this case, additional chemical fixation was used to render the sample completely noninfectious (Maw et al, 2006).…”

Detection of infectious bursal disease virus (IBDV) genome in free-living pigeon and guinea fowl in Africa suggests involvement of wild birds in the epidemiology of IBDV

“…Complementary DNA (cDNA) synthesis Total RNA was isolated from bursa-smeared filter paper using TRIZOL (Invitrogen, Carlsbad, CA, USA) as reported previously [15]. The first strand cDNA was synthesized using Rever Tra Ace reverse transcriptase (RT) (Toyobo, Osaka, Japan) and random primer Pd(N) 6 (Toyobo).…”