Sample preparation for avian and porcine influenza virus cDNA amplification simplified: Boiling vs. conventional RNA extraction.
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.
Friedrich-Loeffler-Institute (FLI), Insel Riems, Germany. Electronic address: sasan.fereidouni@wesca.net.
Sasan R
SR
Fereidouni
Friedrich-Loeffler-Institute (FLI), Insel Riems, Germany.
Elke
E
Starick
Friedrich-Loeffler-Institute (FLI), Insel Riems, Germany.
Mario
M
Ziller
Friedrich-Loeffler-Institute (FLI), Insel Riems, Germany.
Timm C
TC
Harder
International Atomic Energy Agency (IAEA), Vienna, Austria.
Hermann
H
Unger
World Organisation for Animal Health (OIE), Paris, France.
Keith
K
Hamilton
Friedrich-Loeffler-Institute (FLI), Insel Riems, Germany.
Anja
A
Globig
D000482:Allantois / Q000821:virology; D000818:Animals; D001717:Birds; D002988:Cloaca / Q000821:virology; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism*; D005243:Feces / Q000821:virology; D009960:Oropharynx / Q000821:virology; D009975:Orthomyxoviridae / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D013048:Specimen Handling / Q000379:methods*; D013552:Swine
D018076:DNA, Complementary; D012367:RNA, Viral
Influenza viruses; RNA extraction