Sample preparation for avian and porcine influenza virus cDNA amplification simplified: Boiling vs. conventional RNA extraction. 2015

Sasan R Fereidouni, and Elke Starick, and Mario Ziller, and Timm C Harder, and Hermann Unger, and Keith Hamilton, and Anja Globig
Friedrich-Loeffler-Institute (FLI), Insel Riems, Germany. Electronic address: sasan.fereidouni@wesca.net.

RNA extraction and purification is a fundamental step that allows for highly sensitive amplification of specific RNA targets in PCR applications. However, commercial extraction kits that are broadly used because of their robustness and high yield of purified RNA are expensive and labor-intensive. In this study, boiling in distilled water or a commercial lysis buffer of different sample matrices containing avian or porcine influenza viruses was tested as an alternative. Real-time PCR (RTqPCR) for nucleoprotein gene fragment was used as read out. Results were compared with freshly extracted RNA by use of a commercial extraction kit. Different batches of virus containing materials, including diluted virus positive allantoic fluid or cell culture supernatant, and avian faecal, cloacal or oropharyngeal swab samples were used in this study. Simple boiling of samples without any additional purification steps can be used as an alternative RNA preparation method to detect influenza A virus nucleoprotein RNA in oropharyngeal swab samples, allantoic fluid or cell-culture supernatant. The boiling method is not applicable for sample matrices containing faecal material.

UI MeSH Term Description Entries
D009960 Oropharynx The middle portion of the pharynx that lies posterior to the mouth, inferior to the SOFT PALATE, and superior to the base of the tongue and EPIGLOTTIS. It has a digestive function as food passes from the mouth into the oropharynx before entering ESOPHAGUS. Oropharynxs
D009975 Orthomyxoviridae A family of RNA viruses causing INFLUENZA and other respiratory diseases. Orthomyxoviridae includes INFLUENZAVIRUS A; INFLUENZAVIRUS B; INFLUENZAVIRUS C; INFLUENZAVIRUS D; ISAVIRUS; and THOGOTOVIRUS. Influenza Viruses,Myxoviruses,Orthomyxoviruses,Influenza Virus,Myxovirus,Orthomyxovirus
D002988 Cloaca A dilated cavity extended caudally from the hindgut. In adult birds, reptiles, amphibians, and many fishes but few mammals, cloaca is a common chamber into which the digestive, urinary and reproductive tracts discharge their contents. In most mammals, cloaca gives rise to LARGE INTESTINE; URINARY BLADDER; and GENITALIA. Cloacas
D005243 Feces Excrement from the INTESTINES, containing unabsorbed solids, waste products, secretions, and BACTERIA of the DIGESTIVE SYSTEM.
D000482 Allantois An extra-embryonic membranous sac derived from the YOLK SAC of REPTILES; BIRDS; and MAMMALS. It lies between two other extra-embryonic membranes, the AMNION and the CHORION. The allantois serves to store urinary wastes and mediate exchange of gas and nutrients for the developing embryo. Allantoic Membrane,Membrane, Allantoic
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D001717 Birds Warm-blooded VERTEBRATES possessing FEATHERS and belonging to the class Aves. Aves,Bird
D012367 RNA, Viral Ribonucleic acid that makes up the genetic material of viruses. Viral RNA
D013048 Specimen Handling Procedures for collecting, preserving, and transporting of specimens sufficiently stable to provide accurate and precise results suitable for clinical interpretation. Specimen Collection,Collection, Specimen,Collections, Specimen,Handling, Specimen,Handlings, Specimen,Specimen Collections,Specimen Handlings
D013552 Swine Any of various animals that constitute the family Suidae and comprise stout-bodied, short-legged omnivorous mammals with thick skin, usually covered with coarse bristles, a rather long mobile snout, and small tail. Included are the genera Babyrousa, Phacochoerus (wart hogs), and Sus, the latter containing the domestic pig (see SUS SCROFA). Phacochoerus,Pigs,Suidae,Warthogs,Wart Hogs,Hog, Wart,Hogs, Wart,Wart Hog

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