Home » Protocols » CSFV Specific R…
Protocol
CSFV Specific Real Time RT-PCR
Keywords: influenza A, AIV, Newcastle disease viruses, NDV, RT-PCR
Sensitive and specific real-time detection/discrimination of AIV (influenza A) and NDV (Newcastle disease viruses) strains originating from various sources
The AIV/NDV duplex LUX (Light upon extension) assay is qualified for the sensitive and specific real-time detection/discrimination of AIV/NDV strains originating from various sources.
The LUX assay does not require oligonucleotide probe and quencher molecule, instead, it uses only two primers – like conventional PCR –, one of which is labelled with a fluorophore molecule. This set-up enables melting curve analysis on completion of the amplification, which provides a convenient and reliable way for confirming its specificity.
The assay is capable of rapidly detecting a broad range of influenza A and Newcastle disease viruses. Its specificity, sensitivity and cost-efficiency make the assay a useful tool for AI/ND diagnostics.
Required Materials
- QIAamp Viral RNA Mini Kit , for RNA extraction.
-
Primers
- AI-For-37 , forward primer, influenza A
- AI-Rev-181 , reverse primer, influenza A
- ND-703F-JOE , forward primer, Newcastle disease virus
- ND-845R , reverse primer, , Newcastle disease virus
Instructions
-
RNA extraction
RNA is extracted from 140 ?l allantoic fluid, 10% (w/v prepared in distilled water) feces suspensions/organ homogenates or lyophilized virus strains reconstituted in distilled water with the QiAamp Viral RNA Mini Kit (Qiagen, Valencia, CA) as recommended by the manufacturer. RNA is eluted in 60 ?l of elution buffer and one ?l per reaction is used as template for the one-step RT-PCR.
-
Real-time PCR
The Qiagen one-step RT-PCR kit (Qiagen, Valencia, CA) is used for the PCR assays in a final volume of 25 ?l.
The assay set-up is the following;
Name Amount (?l) 5x buffer 5 MgCl2 (25 mM) 1 H2O 12.6 Rnase inhibitor (25 U/?l) 0.2 dNTP (10mM) 1 Forward primer (10 ?M) 0.6 + 0.6 Reverse primer (10 ?M) 1 + 1 Enzyme 1 Sample 1 Melting points are at 88.5 ± 0.5°C 85.5±0.5°C for AIV and NDV, respectively.
Cycling temp. (?C) Time Function Step 50 60 min RT 1 95 15 min activation 2 94 15 sec amplification 3 58 35 sec amplification 4 72 30 sec amplification 5 45 cycles for amplification (step 3-5) 72 1 min 6 In no-template controls distilled water served as target. As internal amplification controls (IAC) pSP73 vector derived (Promega) constructs are used.
In the AIV specific assay, the IAC, which containes plasmid born sequences between the AIV LUX primer binding sites yields a 256 bp PCR product upon amplification. The same type of IAC is used for NDV but it carries a resistance marker flanked by the NDV LUX primer binding sites and yields a 978 bp PCR product. The analytical sensitivity is appr. 20 plasmid copies for both assays.
Reading of fluorescence for FAM and JOE is performed after the synthesis step. Having completed the PCR a melting point analysis is done on both channels.
The assay was tested on the following instruments: iCycler (Bio-Rad, Hercules, CA), ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA), and RotorGene 3000 (Corbett Research, Mortlake, NSW, Australia).
References
-
Application of real-time rt-pcr utilising lux (light upon extension) fluorogenic primer for the rapid detection of avian influenza viruses ,
Author: I. Kiss, P. Germán, L. Sámi, Márta Antal, T. Farkas, G. Kardos, S. Kecskeméti, Á. Dán, S. Belák | Date: December 2006 -
Real-time reverse transcription-polymerase chain reaction detection of Newcastle disease virus using light upon extension fluorogenic primers ,
Author: Márta Antal, Tibor Farkas, Péter Germán, Sándor Belák, István Kiss | Date: 2007