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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Exotic & Emerging Avian Viral Diseases Research » Research » Publications at this Location » Publication #352065

Research Project: Intervention Strategies to Prevent and Control Disease Outbreaks Caused by Emerging Strains of Avian Influenza Viruses

Location: Exotic & Emerging Avian Viral Diseases Research

Title: Armoured exogenous internal control for real-time PCR diagnosis of avian influenza

item ANDREYCHUK, D.B. - Federal Center For Animal Health
item Suarez, David
item ANDRIYASOV, A.V. - Federal Center For Animal Health
item NIKONOVA, Z.B.X - Federal Center For Animal Health
item KOZLOV, A.A. - Federal Center For Animal Health
item CHVALA, IL.A. - Federal Center For Animal Health

Submitted to: Avian Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/14/2019
Publication Date: 7/8/2019
Citation: Andreychuk, D., Suarez, D.L., Andriyasov, A., Nikonova, Z., Kozlov, A., Chvala, I. 2019. Armoured exogenous internal control for real-time PCR diagnosis of avian influenza. Avian Pathology.

Interpretive Summary: The polymerase chain reaction, also known as PCR, is method used to greatly amplify DNA to make it easier to detect. One of the most common applications for PCR is as a diagnostic test to identify pathogens in clinical samples. The use of PCR has become a commonly used laboratory test. One of the concerns with the use of PCR is that some chemicals can inhibit the PCR reaction resulting in a false positive reaction. To identify these false positive reaction a commonly used technique is to include an internal control reaction where DNA from a unique source is added and tested in the clinical sample. This internal control, if positive, provides evidence that the PCR reaction worked correctly and no inhibition is present. This manuscript describes a new and cost effective approach for an Internal control than can easily be procured from commercial companies. This technique was shown to work with a number of different PCR tests, and provides a higher standard for diagnostic testing.

Technical Abstract: An exogenous “armoured” PCR internal control (IC) short RNA was analyzed in conjunction with real-time RT-PCR method for diagnosis of avian influenza. The resistance to nucleases and increased physical stability of the IC was ensured using branched polyethyleneimine (PEI) which was in complex with IC-RNA. The option to add the IC directly to pathological material suspensions allows measurement of the nucleic acids extraction efficiency. Stability of armoured RNA-IC during storage and tissue suspension preparation was shown. The advantage of exogenous “armoured” IC was demonstrated in the experiment with AIV genome detection by qPCR in samples from different species of wild birds. The exogenous IC gave reproducible homogeneous Ct values in all tests.