Abstract
During the 2002-2003 Exotic Newcastle Disease (END) outbreak in Southern California, a high-throughput 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, >0.9999) 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 124-132 |
| Number of pages | 9 |
| Journal | Journal of Veterinary Diagnostic Investigation |
| Volume | 17 |
| Issue number | 2 |
| State | Published - Mar 2005 |
Fingerprint
Keywords
- California
- END
- Exotic newcastle disease
- High-throughput real-time pcr
- RRT-PCR
ASJC Scopus subject areas
- Microbiology
- veterinary(all)
Cite this
High-throughput real-time RT-PCR assay to detect the exotic Newcastle Disease Virus during the California 2002-2003 outbreak. / Crossley, Beate; Hietala, Sharon K.; Shih, Liu Mei; Lee, Lou; Skowronski, Evan W.; Ardans, Alex.
In: Journal of Veterinary Diagnostic Investigation, Vol. 17, No. 2, 03.2005, p. 124-132.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - High-throughput real-time RT-PCR assay to detect the exotic Newcastle Disease Virus during the California 2002-2003 outbreak
AU - Crossley, Beate
AU - Hietala, Sharon K.
AU - Shih, Liu Mei
AU - Lee, Lou
AU - Skowronski, Evan W.
AU - Ardans, Alex
PY - 2005/3
Y1 - 2005/3
N2 - During the 2002-2003 Exotic Newcastle Disease (END) outbreak in Southern California, a high-throughput 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, >0.9999) 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.
AB - During the 2002-2003 Exotic Newcastle Disease (END) outbreak in Southern California, a high-throughput 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, >0.9999) 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.
KW - California
KW - END
KW - Exotic newcastle disease
KW - High-throughput real-time pcr
KW - RRT-PCR
UR - http://www.scopus.com/inward/record.url?scp=15944365908&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=15944365908&partnerID=8YFLogxK
M3 - Article
C2 - 15825492
AN - SCOPUS:15944365908
VL - 17
SP - 124
EP - 132
JO - Journal of Veterinary Diagnostic Investigation
JF - Journal of Veterinary Diagnostic Investigation
SN - 1040-6387
IS - 2
ER -