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)
JVDI, 2014, Vol. 26(1) 72-87
2Proprietary & Confidential
Background
• 2010 AAVLD parasitology committee
• 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
3Proprietary & Confidential
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
4Proprietary & Confidential
Materials and Methods
• Sample collection (Cultured Smegma Samples)• 5 Feeder labs provided 803 samples
• 1 on the West Coast• 1 in the Southwest• 1 in the Central States • 2 in the South
• Each feeder lab ran their own protocol including sample preparation system and real-time PCR
1 Central Study lab (KSVDL) Sample preparation with MagMAXTM Real-time PCR with VetMAXTM T. foetus reagents
5Proprietary & Confidential
Cultured smegma samples provided by feeder labs
Sample Matrix
Source
# of positive
samples *
# of negative samples*
# of inconclusive samples *
Total samples
submitted
Cultured smegma samples
(A) 73 301 0 374
(B) 28 72 0 100
(C) 9 52 2 63
(D) 17 33 0 50
(F) 34 182 0 216
Total 161 640 2 803
* As reported by the feeder labs
6Proprietary & Confidential
Real Time PCR Parameters for Feeder LaboratoriesLab A Lab B Lab C Herds
1-2Lab C Herds
3-6 Lab D Lab F Study LabFinal reaction volume(µL) 20 20 25 25 25 25 25Vol. of T. foetus primer/probe per reaction (µL) 1 1 1 1 1 0.88/1.13-
F&R 1
Volume of extract per reaction(µL) 4 4 8 8 5 5 8Volume Master Mix (µL) 15 15 12.5 12.5 19 18.75 12.5Primer/probe design McMillen McMillen Vet-Max Vet-Max Vet-Max Modified
McMillen Vet-Max
Taq used Universal qPCR
Universal qPCR qPCR MM qPCR MM TaqMan
Univ.MMAbsolute
qPCR low rox mix
qPCR MM
Thermocycler AB 7500 AB 7500 Cepheid SC AB7500 AB7500 AB7500 AB7500Thermocycler mode Standard Standard Fast Standard StandardStage 1 temperature(°C) 95 95 95 95 50/95 95 95Stage 1 time (sec) 10 10 600 10 120/120 15 600Stage2 denaturation (°C) 95 95 95 97 95 95 95Stage 2 denaturation time (sec) 15 15 15 2 20 15 15Stage 2 annealing temp (°C) 55 55 55 55 60 60 55Stage 2 annealing time (sec) 45 45 45 40 45 60 45# cycles 40 40 40 40 40 45 40
Analysis threshold Fixed 2.0 Fixed 2.0Control based threshold-10%
max TF/5% max Xeno
Control based threshold-10%
max TF/5% max Xeno
Fixed 0.2 Control based threshold- 10% max TF/Xeno
Analysis baseline setting 3-15 3-15 autoPositive (Ct) <35 <35 <36 <36 <38 <37 <38
Suspect/Inconclusive(CT) 35-40 35-40 36-40 36-40 38-39 >37 38-40/Xeno 28.5-31.5
Negative(CT) >40 >40 >40 >40 >40 undetected Undetected/Xeno 28.5-31.5
Internal extraction control No No Yes <36 CT Yes <36 CT No Yes Yes CT = 28.5-31.5
7Proprietary & Confidential
Results: Individual Sample Testing
Study Lab Result / Feeder Lab Result
Sample Matrix
Lab Sourc
e
Sample preparati
on system
Pos/Pos
Pos/Neg
Pos/Inc
PresPos/Neg
Neg/Pos
Neg/Inc Neg/Neg Total
Percent agreemen
t
Cultured smegma samples
A Boiling 71 5 0 1 2 0 295 374 97.9B Boiling 21 10 0 0 7 0 62 100 83.0C MagMax 9 2 2 0 0 0 50 63 96.7D Boiling 17 4 0 1 0 0 28 50 90.0F Qiagen 34 0 0 1 0 0 181 21 6 99.5
Results All labs Multiple 152 21 2 3 9 0 616 803 95.6
Order of the call = KSVDL Study Lab / Feeder LaboratoryPos = positive, Neg = negative, Inc = inconclusive, PresPos= presumptive positive
Study Lab Results vs. Feeder Lab Results
8Proprietary & Confidential
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.
9Proprietary & Confidential
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
10Proprietary & Confidential
Pooling results
Sample ID*
Individual test CT
Pooled1:5 Test CT
Pooled1:3 Test CT
Study lab final call
Pooled 1:5 call
Pooled 1:3 call
1 C-6-11 35.05 Undetected Undetected Positive Negative Negative2 A-40-5 35.20 37.83 37.86 Positive Positive Positive3 A-39-2 35.41 35.93 34.82 Positive Positive Positive4 F-1-7 35.43 35.43 35.53 Positive Positive Positive5 F-18-1 35.52 36.16 34.75 Positive Positive Positive6 C-1-23 35.93 35.75 35.82 Positive Positive Positive7 B-8-4 36.02 34.91 35.59 Positive Positive Positive8 F-19-10 36.30 33.48 32.82 Positive Positive Positive
10 A-27-9 36.36 Undetected Undetected Positive Negative Negative9 B-4-1 36.45 Undetected Undetected Positive Negative Negative
11 C-6-10 37.20 Undetected 37.69 Positive Negative Positive12 A-41-2 37.89 36.92 Not tested Positive Positive Not tested13 C-4-5** 38.77 (ave of 4) Undetected Undetected Positive WFA* Negative Negative14 C-6-15** 39.09 (ave of 3) Undetected Undetected Positive WFA Negative Negative
15 A-24-10** 39.12 (ave of 2) Undetected Undetected Suspect Positive WFA Negative Negative
Effect of pooling for T. foetus samples with CT>35 after individual testing
*WFA: Suspect workflow A; ** samples confirmed T. foetus by sequencing
11Proprietary & Confidential
Pooling results
1:5 Pools• 1:5 pooling of positive samples with a CT of 35 and below were all
detected• Only 3 of 9 positive samples with CTs between 36-39.9 were detected
in 1:5 pools• Pooling at 1:5 missed 4% (7/176) of T. foetus positive samples
1:3 Pools• Only 8 of 15 positive samples with CTs between 36-39.9 were
detected in the 1:3 pools• 1:3 pooling missed 3.5% (6/176) of T. foetus positive samples
12Proprietary & Confidential
Sequencing primer design for nested PCR
(R-TFSM-primer)
(O-F-TFSM-Primer) (M13-I-F-TFSM-Primer)
(M-13-R-TFSM-primer)
TTAGCTTTCTTT GCGA T. foetus
TTAGCTAACAAT GCGA S. moskowitzi
Primers for Nested PCR Abbreviation Primer SequenceForward outer forward primer (O-F-TFSM-Primer) CCTTAGGCAATGGATGTCTTGGC
Reverse primer (R-TFSM-primer) GCGCAATGTGCATTCAAAG
M13 Forward Inner primer (M13-I-F-TFSM-Primer)
TGTAAAACGACGGCCAGTCTTACACGATGAAGAACGTTGC
M13 Reverse primer (M-13-R-TFSM-primer)
CAGGAAACAGCTATGACCGCGCAATGTGCATTCAAAG
GenBank: GQ254636.1 Simplicimonas moskowitziGenBank: AY349189.1 Tritrichomonas foetus
13Proprietary & Confidential
Sequencing results for 175 T. foetus positives
175/176 T. foetus positive samples, including three late risers, were confirmed T. foetus by DNA sequencing
14Proprietary & Confidential
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
15Proprietary & Confidential
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
16Proprietary & Confidential
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
17Proprietary & Confidential
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