High-Throughput Real-Time RT-PCR Assay to Detect the Exotic Newcastle Disease Virus during the California 2002–2003 Outbreak

Abstract

Abstract. During the 2002During the -2003 Exotic Newcastle Disease (END) outbreak in Southern California, a highthroughput real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) system was developed to respond to the large diagnostic and surveillance sample workload. A 96-well RNA extraction method, using magnetic bead technology, combined with a 96-well RRT-PCR assay, allowed 1 technician to process and test more than 400 samples per day. A 3-technician team could complete testing on approximately 1,900 samples per day. The diagnostic sensitivity of the high-throughput RRT-PCR assay was 0.9967 (95% CI 0.9937-0.9997) based on 926 virus isolation confirmed positive samples. Diagnostic specificity using an initial 434 virus isolation confirmed negative samples was 100%. A diagnostic specificity of 0.9999 (95% CI 0.9999, Ͼ0.9999) was subsequently calculated on the basis of 2 false-positive results among 65,343 surveillance samples collected after the final END-positive case was confirmed in May 2003. Assay performance over 500 replicates, including reproducibility of the combined extraction and RRT-PCR amplification steps yielded a standard deviation of 0.70 RRT-PCR cycle thresholds (Ct) and a standard deviation of 0.59 Ct for the RRT-PCR steps alone. The high-throughput RRT-PCR developed for END contributed significantly to the 2002-2003 END control effort, reducing the predicted timeline for eradication from 3 years to just 11 months, primarily because of the large number of samples that could be rapidly tested. The 96-well approach described for high-throughput END RRT-PCR could be adapted to other rapid, high-volume testing needs, as required for potential foreign animal disease responses or intensive surveillance efforts.

Environmental Air Sampling to Detect Exotic Newcastle Disease Virus in Two California Commercial Poultry Flocks

“…Virus was also detected by RRT-PCR and virus isolation in oropharyngeal swabs and from pooled tissue. The virus detected was confirmed as END virus by direct sequence analysis of RRT-PCR amplicons 3,4 and represented the first time during the outbreak that the fusion protein cleavage site consisting of the amino acid pattern 110 GGRRQRRFVG 119 (GenBank AY266476) was detected. The fusion protein cleavage site sequence varied from the index case sequence of 110 GGRRQKR FVG 119 (GenBank AY216490) by 1 amino acid and was subsequently detected from commercial chickens, noncommercial chickens, and domestic pigeons.…”

“…The finding was not unexpected because virus isolation using egg inoculation was found to be 1-10 EID 50 more sensitive compared with the RRT-PCR used in this study. 4 Sequence confirmation for END RRT-PCR amplicons from END virus-positive samples from flock B confirmed END virus with the variant fusion protein cleavage site sequence. Time zero for both the air-sampled flocks as well as 0-, 2-, and 4-hour samples collected in a poultry-free environment subsequent to flock sampling were negative for END virus by both RRT-PCR and virus isolation.…”

“…The RRT-PCR assay targeted the END fusion protein cleavage site and has a reported sensitivity of 99.67% and specificity 99.997% for swab samples and an analytical detection limit between 10 and 100 egg infective doses (EID) 50 for END virus. 4 Oropharyngeal swabs and tissue samples from 5 randomly selected apparently healthy chickens were also obtained from each flock within 24 hours of airsample collection.…”

“…The collection fluid was recycled in the system 6-20 times/minute for an estimated 50,000/minute concentration ratio and evaporated fluid was replaced automatically from a 150-ml reservoir. Subsamples (2 ml) were removed from the collector at 0, 2, and 8 hours and transported on ice to the California Animal Health and Food Safety Laboratory System (CAHFS) for standard egg-inoculation virus isolation, RRT-PCR, and PCR-amplicon sequence analysis, as described previously, 4 with modification to a single-tube format using a Smartcycler b PCR instrument. The RRT-PCR assay targeted the END fusion protein cleavage site and has a reported sensitivity of 99.67% and specificity 99.997% for swab samples and an analytical detection limit between 10 and 100 egg infective doses (EID) 50 for END virus.…”

“…During the 2002-2003 END control campaign, more than 80,000 samples were tested by real-time reverse transcriptase PCR (RRT-PCR) using primarily oropharyngeal, tracheal, and cloacal swabs, plus a smaller number of tissue and environmental swab samples. 4 Swab samples were collected from individual birds obtained through custom slaughter surveillance, routine flock-mortality surveillance, and live-bird testing. Live-bird testing in both commercial and noncommercial flocks used swab specimens collected from a statistically determined representative sample of the population, typically between 10 and 79 birds depending on the flock size.…”