Rapid and Simultaneous Detection of Avian Influenza and Newcastle Disease Viruses by Duplex Polymerase Chain Reaction Assay

Virus isolates

Virus strains and bacteria used in the specificity assay of duplex reverse transcription-polymerase chain reaction (dRT-PCR) are listed in Table 1. NDV strains B1, La Sota (Phylavac^TM, Vitapest^TM), V4 Queensland and AIV strain A/tk/Turkey/8/11/05 were kindly provided by Ceva-Phylaxia (Budapest, Hungary) and the Central Veterinary Institute (CVI, Budapest, Hungary), respectively. Other AIV/NDV field isolates were collected from clinical samples in the Virology Department of the CVI, Debrecen, Hungary, in the period of 1969–1990. Virus samples were identified by propagation in embryonated chicken eggs and haemagglutination activity (HA) test according to the OIE manuals (Alexander, 2004).

Determination of virus titre

Virus stock solution was diluted 1 : 10 in sterile PBS supplemented with 100 U/ml of penicillin-G and 0.1 mg/ml of streptomycin (Sigma, St Louis, MO, USA). Embryonated chicken eggs were inoculated with 0.2 ml diluted virus preparation on the 10th day of incubation by the allantoic sac route (five eggs/dilution). Between days 3 and 6 post-inoculation allantoic fluid was collected and tested for haemagglutination with 0.5% chicken red blood cells (CRBCs) in PBS. The virus titre was determined by the Reed-Muench method (Reed and Muench, 1938).

RNA extraction and cDNA synthesis

RNA was extracted from 140 μl volume of sample (allantoic fluid/lyophilized virus stock reconstituted in distilled water/suspended stool sample) by using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) as recommended by the manufacturer. The reverse transcription (RT) of RNA to cDNA was performed using Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Invitrogen, Carlsbad, NM, USA), pd(N)6 random hexamer 5′-phosphate and RNAguard (Amersham, Piscataway, NJ, USA) according to the manufacturer's instructions.

Primer design and polymerase chain reaction

Avian influenza virus and NDV specific oligonucleotide primers were designed by using nucleotide sequences available in GenBank, the software package DNASTAR and the program Primer Designer Version 2.0 (Scientific and Educational Software).

Primer pair AIV-MP37-For (5′-GATGAGTCTTCTAACCGAGG-3′) and AIV-MP180-Rev (5′-GTCTTTAGCCAYTCCATGAG-3′) amplifies a 144 bp long fragment from the AIV matrix protein gene (M), while primers NDVF-for2 (5′-TGGGAAGATGCAGCARTTTG-3′) and NDVF-rev1 (5′-RCCCAAGAGTTGAGTCTGTGA-3′) are specific for the fusion protein gene (F) of NDV and the size of their amplification product is 316 bp (Y: pyrimidine; R: purine).

The optimization of PCR conditions was performed using the cDNA from AIV strain A/gf/Hu/2/75 (H7) and NDV vaccine strain La Sota as template in the MyCycler (Bio-Rad, Budapest, Hungary) and the RapidCycler (Idaho Technology, Idaho Falls, ID, USA) devices. Optimal PCR conditions are as follows:

Amplification in the MyCycler instrument was performed in a final volume of 20 μl, the reaction mix consisted of 2 μl AmpliTaq Gold 10x PCR buffer, 3.2 μl 25 mm MgCl₂, 0.1 μl AmpliTaq Gold polymerase (5 U/μl; Applied Biosystems, Foster City, CA, USA), 0.4 μl 10 mm dNTP blend (0.2 mm final concentration of each), 1 μl 5 mg/ml BSA (Promega, Madison, WI, USA), 0.2–0.2 μl of primers AIV-MP37-For; AIV-MP180-Rev; NDVF-for2 and NDVF-rev1 (25 pmol/μl; Biomi, Gödöllő, Hungary), 1 μl template cDNA and 11.9 μl PCR grade H₂O (Life Technologies, Gaithersburg, MD, USA). The PCR cycling program was: 95°C 7 min; 40 cycles of 95°C 30 s, 58°C 30 s, 72°C 60 s; followed by 72°C 7 min.

Amplification in the RapidCycler was carried out as described for MyCycler. The total reaction volume was 10 μl and the quantity of PCR components was reduced accordingly, except that 1 μl 5 mg/ml BSA; 0.1 μl AmpliTaq Gold polymerase and 1 μl template cDNA was added to the reaction. The thermal profile was: 95°C 7 min; 40 cycles of 94°C 20 s, 58°C 20 s, 72°C 20 s; followed by 72°C 7 min. PCR grade water was used in the reactions as negative control.

DNA sequencing and nucleotide sequence accession numbers

The amplified DNA fragments were purified with the QIAquick PCR purification kit (Qiagen) according to the manufacturer's instructions. Double-stranded sequencing reaction was performed by using the BigDye Terminator Cycle Sequencing v3.1 kit (Applied Biosystems) and the same primers as in the dRT-PCR. Samples were analysed in an automated sequencer ABI3100 (Biomi). The nucleotide sequences of four AIV and four NDV amplicons have been deposited in GenBank (Acc. No.: DQ533836-39 and DQ455008-455011).

PCR specificity and sensitivity assay

To determine the specificity of the dRT-PCR assay, RNA/DNA was extracted from AIV/NDV strains and 16 heterologous avian pathogens (Table 1) by using QIAamp Viral RNA Mini Kit and DNeasy Tissue Kit (Qiagen). Nucleic acid samples were processed and tested as described above.

The sensitivity of the dRT-PCR assay was determined by using AIV/NDV stock solutions with known egg infectious dose (EID)₅₀ values (AIV strain A/gf/Hu/2/75: 10^+7.5 EID₅₀/0.2 ml and NDV strain La Sota: 10^+9.2 EID₅₀/0.2 ml). Serial 10-fold dilutions were prepared of the AIV/NDV stock solutions with sterile H₂O, then RNA was extracted and cDNA was synthesized from each dilution as described above. First, the detection limit was measured separately for AIV and NDV by adding only AIV or NDV cDNA to the reaction (10¹–10⁸× dilutions). To determine the ability of the dRT-PCR to detect and differentiate AIV/NDV in the same reaction, different dilutions of AIV and NDV cDNAs were mixed in various combinations (in 1 : 1 ratio) and the mixture was used as template. In the first set of experiment, AIV and NDV stock solutions were diluted in parallel (10¹–10⁸× dilutions, Fig. 1a), while in the second set the titre of one virus was constant (10³–10⁴× dilutions) and the quantity of the other virus was changing (10¹–10⁸× dilutions, Fig. 1b).

Detection of AIV/NDV from experimentally infected stool specimens

Chicken stool samples previously giving negative test results in the dRT-PCR were collected and pooled. To 0.2 g pooled faeces 50 μl of the 10¹–10⁸× dilutions of AIV (A/gf/Hu/2/75) or NDV (La Sota) stock solutions was added, then samples were suspended in 450 μl sterile H₂O, thoroughly vortexed and centrifuged (2 min; 4700 g; RT). Viral RNA was extracted from the supernatant and processed as described above. The 10-fold dilution applied during the preparation of samples was calculated in the results.

Detection of AIV/NDV in stool samples from vaccinated chickens

A breeder flock was vaccinated with Avinew (Merial, Lyon, France) at day 1 and with TAD ND vac HB1 (Lohmann, Cuxhaven, Germany) at 21 days of age. Vaccines contained VG/GA and Hitchner B1 live NDV strains, respectively. Control chickens were left untreated and kept separately. After 2 weeks of the second vaccination, stool specimens were collected and pooled according to the EC Regulation No. 1003/2005 (CEC, 2005). To 0.2 g pooled faeces 0.5 ml sterile H₂O was added, then samples were thoroughly vortexed and centrifuged (2 min; 4700 g; RT). Viral RNA was extracted from the supernatant and processed as described above. Results were confirmed by a PCR assay using universal and pathotype specific primer pairs able to differentiate lentogenic and velogenic NDV strains (Kant et al., 1997). In order to examine the effect of double infection on the sensitivity of dRT-PCR serial, 10-fold dilutions of AIV stock solution were made and aliquotes were added to the pooled stool samples collected from vaccinated birds. Viral RNA was extracted, processed and tested in dRT-PCR as described above (see details of spiking in previous section). The 10-fold dilution applied during the preparation of spiked stool samples was calculated in the results.

Duplex RT-PCR identification of AI and ND viruses in different thermocyclers

The nucleotide sequences of known AIV and NDV isolates were compared in multiple alignments and primers targeting highly conserved regions, the matrix protein gene (M) of AIV and the fusion protein gene (F) of NDV, were designed. The optimal conditions for the duplex PCR assay were determined in two thermal cyclers using the cDNA from AIV and NDV as template. A conventional heat block thermal cycler, MyCycler, was tested to assure that the dRT-PCR can be applied on the currently most prevalent system. In the RapidCycler, cycling system is based on the heat transfer by ventilating hot/cool air to the samples. Rapid heat exchange, the use of 1.5 mm glass reaction cuvettes (capillaries), small reaction mix volume (10 μl) allows the use of relatively short cycling times. Optimal reaction conditions were almost identical in the two instruments, there were notable exceptions only in the total volume of reaction mix (MyCycler: 20 μl; RapidCycler: 10 μl), cycling times (MyCycler: 30–30–60 s; RapidCycler: 20–20–20 s) and BSA concentration (MyCycler: 0.25 mg/ml; RapidCycler: 0.5 mg/ml).

Specificity of duplex RT-PCR assay

In the first step, to examine the specificity of the dRT-PCR assay, 13 AIV isolates (subtypes: H4; H5; H6; H7; H10; H11), 16 NDV isolates (nine lentogenic and seven velogenic strains) and three NDV vaccine strains were subjected to dRT-PCR, as shown in Table 1. The expected PCR products of 144 bp and 316 bp length, respectively, were amplified from all AIV/NDV samples. In the second step, the specificity was further investigated by testing other potential avian pathogens, including bacteria. No cross-reaction was observed with the heterologous disease-producing agents. The identity of the 144 bp (AIV) and 316 bp (NDV) PCR products was confirmed by means of DNA sequence analysis and comparative search against GenBank.

Sensitivity of dRT-PCR assay

The sensitivity of the dRT-PCR assay was determined using serial 10-fold dilutions of AIV and NDV stock solutions. The detection limit of the duplex PCR was approximately 10^+0.5 EID₅₀/0.2 ml for AIV and 10^+2.2 EID₅₀/0.2 ml for NDV when cDNA templates were added separately to the reaction (data not shown). No alteration was observed in the sensitivity when both target cDNAs (AIV and NDV) were present in the PCR mix and they were diluted in parallel (Fig. 1a). However, when one viral cDNA was added to the reaction in excess a slight decrease (maximum one order of magnitude) was observed in the detection limit (Fig. 1b).

The type of the thermal cycler did not influence the endpoint of detection, as we had identical results in the MyCycler and in the RapidCycler.

Detection of AIV/NDV from experimentally infected stool specimens

We evaluated the ability of dRT-PCR to amplify the corresponding genomic regions from viral RNA extracted from faecal samples. Chicken stool samples were experimentally infected with a dilution series of AIV or NDV stock solutions. The detection limit of duplex RT-PCR was approximately 10^+0.5 EID₅₀/0.2 ml for AIV and 10^+2.2 EID₅₀/0.2 ml for NDV in this reaction. The assay was able to detect the corresponding virus(es) from each specimen with the same sensitivity as from allantoic fluid, in spite of the fact that stool samples are reported to contain several PCR inhibitory compounds (bile salts, haemoglobin degradation products, etc.) that seriously decrease the efficiency of nucleic acid detection (Higuchi, 1989; Radstrom et al., 2004).

Detection of AIV/NDV in stool samples from vaccinated chickens

Stool samples collected from a chicken flock previously inoculated with NDV vaccine strains were all positive by dRT-PCR for the presence of NDV, while no amplification product was observed in the negative controls. Positive samples were subjected to a pathotype specific PCR (Kant et al., 1997) and the results proved that specimens contained lentogenic NDV RNA (data not shown).

When the effect of double infection was investigated on the sensitivity of dRT-PCR by the addition of serial 10-fold dilutions of AIV stock solutions to the stool samples collected from vaccinated birds, NDV remained detectable in all samples, though high concentration of AIV was sometimes present in the reaction (Fig. 1c, lane 1–2: 10^+5.5–10^+4.5 EID₅₀/0.2 ml). The endpoint of detection for AIV was calculated as approximately 10^+1.5 EID₅₀/0.2 ml.