1 Proprietary & Confidential The world leader in serving science Proprietary & Confidential Pooling of cultured samples and comparison of multistate laboratory workflows with the MagMAX sample preparation system and VetMAX quantitative polymerase chain reaction reagents for detection of Tritrichomonas foetus– colonized bulls Lee Effinger Lalitha Peddireddi Marilyn Simunich Richard Oberst Catherine O’Connell Ivan Leyva-Baca Oregon Department of Agriculture, Animal Health and Identification Division, Animal Health Laboratory, Salem, OR (Effinger) Department of Diagnostic Medicine/Pathobiology (Peddireddi), Kansas State University, Manhattan, KS Kansas State Veterinary Diagnostic Laboratory (Oberst), Kansas State University, Manhattan, KS Animal Health Laboratory, Idaho State Department of Agriculture, Boise, ID (Simunich) Animal Health and Food Safety Group at Life Technologies, Austin, TX (Leyva-Baca, O’Connell) JVDI, 2014, Vol. 26(1) 72-87
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Lee Effinger Lalitha Peddireddi Marilyn Simunich Richard Oberst Catherine O’Connell
Pooling of cultured samples and comparison of multistate laboratory workflows with the MagMAX sample preparation system and VetMAX quantitative polymerase chain reaction reagents for detection of Tritrichomonas foetus –colonized bulls. Lee Effinger Lalitha Peddireddi Marilyn Simunich - PowerPoint PPT Presentation
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1Proprietary & ConfidentialThe world leader in serving scienceProprietary & Confidential
Pooling of cultured samples and comparison of multistate laboratory workflows with the MagMAX sample preparation system and VetMAX quantitative polymerase chain reaction reagents for detection of Tritrichomonas foetus–colonized bullsLee EffingerLalitha PeddireddiMarilyn SimunichRichard OberstCatherine O’ConnellIvan Leyva-Baca
Oregon Department of Agriculture, Animal Health and Identification Division, Animal Health Laboratory, Salem, OR (Effinger) Department of Diagnostic Medicine/Pathobiology (Peddireddi), Kansas State University, Manhattan, KS Kansas State Veterinary Diagnostic Laboratory (Oberst), Kansas State University, Manhattan, KS Animal Health Laboratory, Idaho State Department of Agriculture, Boise, ID (Simunich) Animal Health and Food Safety Group at Life Technologies, Austin, TX (Leyva-Baca, O’Connell)
• Proposed a study to determine whether T. foetus samples can be pooled in order to reduce the costs for testing
• Lee Effinger from Oregon State Department of Agriculture led Experimental Design for the project
• Marilyn Simunich Idaho State Department of Agriculture served as Study Coordinator & Data Keeper
• The Life Technologies Animal Health & Food Safety Group agreed to support the study
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Objectives
1. Determine the effect of pooling a single positive sample having various CT ranges with four negative samples (1:5). If a negative effect was seen, a 1:3 pooling study would then be conducted
2. Compare different sample preparation systems and various real-time PCR (feeder lab workflows) with the 5X MagMAXTM-pathogen RNA/DNA purification kit and amplification with VetMAXTM T. foetus reagents (Life Technologies workflow)
3. Assess the specificity of the VetMAXTM T. foetus reagents by sequencing all positive samples with CT values less than 38 and suspect sample CT values between 38 and less than 40 cycles
Order of the call = KSVDL Study Lab / Feeder LaboratoryPos = positive, Neg = negative, Inc = inconclusive, PresPos= presumptive positive
Study Lab Results vs. Feeder Lab Results
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Conclusions Individual Testing
• 803 smegma samples were provided by feeder labs (FL)
• All the samples were tested by study laboratory with Life Technologies workflow systems:• MagMAXTM
• VetMAXTM T. foetus reagents
• Agreement of 95.6% was reached with 768/803 samples between feeder labs and study lab
• Interestingly, Lab F reached almost 100% agreement using a different sample prep system and a modified McMillen’s assay
• Study laboratory (KSVDL) with LT protocol identified 24 more positives than the feeder laboratories. On retesting, one of the feeder labs missed 9 samples reported as positives.
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Pooling Study
Laboratory ID
Positive Samples Available
Negative Samples Available
Negatives Needed
Deficit /Surplus of Negative Samples
A 77 297 308 -11 B 31 69 124 -55 C 13 50 52 -2D 21 28 84 -56F 34 181 136 +45Total 176 625 704
Positives, presumptive positive and negative samples used from each lab for pooling
175/176 T. foetus positive samples, including three late risers, were confirmed T. foetus by DNA sequencing
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Sensitivity, specificity, & predictive values of positive & negative results for all cultured smegma samples
Calculation Formula Result Result
% Sensitivity True Positives
True Positives + False Negatives X 100
175_ 175 + 0 x100
100%
% Specificity True Negatives
True Negatives + False Positives X 100
625___ 625 + 3 x100
99.52%
Predictive value of a
positive test
True Positives
True Positives + False Positives X 100
175__ 175 + 3 x100
98.31%
Predictive value of a
negative test
True Negatives
True Negatives + False Negatives X 100
625__ 625 + 0 x100
100%
* Calculations made after qPCR and sequence confirmation
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Sequencing Results
• 175/176 positive samples by qPCR were able to be sequenced
• 1 sample (A-7-25) with a CT 33.95 was not able to be sequenced. • It is possible that there are point mutations in this positive sample in the
sequencing primer regions, which were designed based on a few T. foetus and a single S. moskowitzi sequences from GenBank
• Most importantly, none of the samples reported S. moskowitzi DNA sequences
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Overall results
• 95.6 % agreement was reached between Study Lab (KSVDL) using Life technologies MagMAXTM and VetMAXTM T. foetus reagents and the feeder laboratories
• 1:5 Pooling it is likely to miss 4% of the positives• 1:3 Pooling it is likely to miss 3.5% of the positives
• DNA sequencing• 175/176 positive samples were confirmed to be T. foetus, the 176th
sample could not be sequenced with the primers designed for this study
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AcknowledgementsLalitha Peddireddi, KSVDL – performed the study at KSVDLLee Effinger, ODA-Animal Health LaboratoryMarilyn Simunich, Idaho State Dept. of AgricultureCate O’Connell, Life TechnologiesMangkey Bounpheng, Texas Veterinary Medical Diagnostic LaboratoryDawn Bueschel, NMDA Veterinary Diagnostic ServicesMuthu Chengappa, Kansas State Veterinary Diagnostic LaboratoryAlfonso Clavijo, Texas Veterinary Medical Diagnostic LaboratoryKris A. Clothier, California Animal Health & Food Safety Lab SystemHemant K. Naikare, Texas Veterinary Medical Diagnostics LaboratoryJeff Zinza, Life TechnologiesMary Anne Williams, Life Technologies