1

1

Guidelines for Development and Performance of PCR 2

Assays in Veterinary Diagnostic Laboratories 3

4

Laboratory Technology Committee 5

May 1, 2013 6

Members and affiliations: 7

• Beate Crossley CAHFS co-chair 8

• Kathy Toohey-Kurth WVDL co-chair 9

• Amy Glaser, AHDC Cornell 10

• Jan Pedersen, NVSL 11

• Monica Reising, CVB 12

• Deep Tewari, PA Dept of Ag 13

• Susanne Hinkley, GeneSeek NE 14

• Steve Bolin, Michigan State 15

• Mangkey Bounpheng, TDVML 16

• Suelee Robbe-Austerman, NVSL 17

• Beth Harris, NVSL 18

• Donna Mulrooney, Oregon State Univ 19

• Roger Maes, Michigan State 20

• Roman Pogranichniy, ADDL Purdue 21

• Shri Singh, Murray State University KY 22

• NAHLN – MTWG subcommittee 23

2

De Novo PCR assay Development 1

Guidelines for the Performance Evaluation Process 2

3

Development of novel PCR-based assays is often required of laboratories for a variety of 4

applications. The guidelines below are meant to provide a template to assist the development 5

process from initial assessment of need and intended use to implementation. No attempt has been 6

made to provide performance benchmarks, as assays are required for a wide variety of needs, 7

from detection of nucleic acid in clinical samples to typing of bacterial or viral isolates. It is the 8

responsibility of the individual developing the assay to establish acceptable assay characteristics 9

fit for the intended use of the assay. Use of these guidelines will facilitate evaluation of assay 10

performance between labs and allow independent assessment and comparison of assay 11

characteristics when published. This document is not intended to be a review of assay validation 12

principles. A comprehensive discussion of assay validation can be found in references provided 13

at the end of this document. 14

15

1. Assay Justification 16 1.1. Introduction 17

1.1.a. Why assay is needed 18

1.1.b. Literature review of topic/pathogen (check box), stored/archived 19

1.1.c. Description of choice of target gene(s)/sequence(s) 20

1.1.d. Choice of Gold Standard and Justification 21

1.1.e. Description of Controls and Standards 22

23

1.2. Scope and Application (description of intended use) 24

1.3. Assay Design 25

1.3.a. Conventional or real time detection 26

1.3.b. Materials and equipment needed 27

28

1.4. Assay feasibility 29

1.4.a. In silico evaluation 30

- Appropriate primer and probe sites in target region 31

- BLAST evaluation of primer sequences and primer specificity 32

33

2. Initial assessment of primer and probe sequences 34 2.1. Standard curve using 10-fold serial dilutions of target sequence 35

2.1.a. During the feasibility stage 36

i. It is recommended to test at least 2-5 primer pairs 37

ii. Use buffer diluents to compare initial primer performance. 38

iii. Assess performance with dilutions of target in nucleic acid from appropriate 39

sample matrix at an early point in development. 40

iv. Some labs use isolates, other labs use a synthetic construct. If using a synthetic 41

construct, it is advised to test full length genomic length target at an early point in 42

development in order to assess the effect of secondary structure. 43

2.1.b. Include dilutions that facilitate determination of assay limits/analytical sensitivity 44

and cover expected concentrations in clinical samples. 45

3

2.1.c. Dilutions should extend two 10-fold dilutions beyond detection limit. Smaller 1

dilutions can be used to determine endpoint but many labs find little value added 2

for the extra cost incurred. 3

4

5

3. Description of Assay Optimization 6 3.1. Optimization of reagents. If assay initially does not meet required performance 7

characteristics, optimization of reaction conditions can be attempted. Suggested 8

optimization adjustments: 9

3.1.a. Primer annealing temperature 10

3.1.b. Primer sequence/probe sequence corrections 11

3.1.c. Buffer adjustments (MgCl2, BSA, etc), different commercial 2x kits 12

3.1.d. Cycling parameters 13

14

If the PCR conditions can be adjusted to meet assay performance requirements, then 15

validation is continued as outlined below. If the assay performance is not acceptable, re-16

design of primers is required. Alternatively, if the laboratory is trying to develop conforming 17

assays that all run with identical buffers, enzyme, and cycling conditions, then optimization is 18

counter-productive and re-design should be pursued. 19

20

Once assay characteristics appear to be acceptable, feasibility should also include testing of 21

the assay across expected strains and matrices (inclusivity) and also against near genetic 22

neighbors and similar disease agents to ensure there are no cross reactions (exclusivity). 23

Performing these steps during the feasibility stage will avoid spending time and money to 24

characterize an assay that will ultimately fail to be useful. 25

26

4. Evaluation of Assay Performance: 27

28 A series of experiments using known concentrations of target sequence (for example virus titer, 29

PCR amplicon, CFUs, copy number controls) in the most common sample matrix for that 30

analyte is used to determine the assay characteristics described below. Concentrations of target 31

should span the range expected in clinical samples and include 2 dilutions beyond the expected 32

limit of detection. A minimum of three independent dilution series are tested using a unique 33

master mix for each series and preferably tested on separate days. Concentrations should 34

encompass the expected amount of analyte in field samples. If possible, include concentrations 35

that exceed the upper and lower limits of detection. The results will be used to determine the 36

following assay characteristics: 37

38

4.1. Range and Linearity 39 4.1.a. Linear regression analysis log concentration vs Ct. 40

4.1.b. Calculate slope, linearity (R2), and efficiency ({10

(-1/slope)}-1). 41

42

4.2. Limit of Detection (LOD) 43 Determination of LOD requires a known concentration of analyte. LOD determinations 44

should be performed using nucleic acids derived from titered stocks of viruses or 45

bacteria, artificial phage, amplicons or plasmid constructs containing the target sequence. 46

4

The target may be in sample matrix. A more precise LOD can be determined if copy 1

numbers are calculated from purified single molecules of nucleic acid of known 2

molecular weight. All options have advantages and disadvantages. 3

4

Limit of detection must be empirically determined for each pathogen strain or variant 5

(e.g. BVDV 1a, 1b and 2). Approved reference strains or thoroughly characterized 6

isolates or positive samples should be used. 7

8

4.3. Precision 9 Precision should be established through repetitively testing replicates of the target 10

sequence at three concentrations, high, medium, and low. Guidelines developed for assay 11

comparison can be used. A series of dilutions are prepared for a single reference strain 12

sufficient for the entire series of experiments. Each concentration is tested 5 times within 13

each of 6 independent runs. 14

4.3.a. Repeatability (within-run imprecision) 15

Analysis will be provided in the Methods Comparison publication. 16

17

4.3.b. Reproducibility (between-run imprecision) 18

Analysis will be provided in the Methods Comparison publication. 19

20

21

4.7. Diagnostic Performance 22 4.7.a. Test a series of samples previously tested by another method. It is recommended 23

to test 30 positive samples with different concentrations of target and 30 negative 24

samples. 25

4.7.b. Secondary assays to confirm specificity/target sequence amplification might be 26

employed; they will only confirm specificity. 27

4.7.c. Refer to OIE guidelines for sample size determination for de Novo testing. 28

29

5. Conclusions Regarding Assay Validation 30 Does the assay function adequately for the purpose intended? 31

32

6. Ongoing assessment of assay function 33 It is prudent to perform in silico analysis of primer sequences to ensure that your assay can 34

detect emerging isolates and sequence variants as needed according to the rate of change 35

of your target as appropriate. 36

37

7. Re-validation 38 Note that changes in the validated assay such as chemistry, platform, species and matrix all 39

require a methods comparison (Reising et al) 40

41

42

43

44

45

46

5

7. References: 1

2 Jacobsen, R (2010) 3

Principles and Methods of Validation of Diagnostic Assays for Infectious Diseases. 4

OIE Terrestrial Manual Chapter 1.1.4/5. 5

6

Burd, E.M. (2010) 7

Validation of Laboratory-Developed Molecular Assays for Infectious Diseases. 8

Clinical Microbiology Reviews 23:550-575. 9

10

Bustin, SA; Benes,V; Garson, JA; Hellemans, J; Huggett, J; Kubista, M; Mueller, R; Nolan, T; 11

Pfaffl, MW; Shipley, GL; Vandesompele, J; and Wittwer, CT (2009) 12

The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR 13

Experiments. 14

Clinical Chemistry 55 p. 611-22. 15

16

Bustin SA, Beaulieu JF, Huggett J, Jaggi R, Kibenge FS, Olsvik PA, Penning LC, Toegel 17

S.(2010) 18

MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based 19

quantitative real-time PCR experiments. 20

BMC Mol Biol. 21:71-74. 21

22

Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl 23

MW, Shipley GL, Vandesompele J, Wittwer CT. (2011) 24

Primer sequence disclosure: a clarification of the MIQE guidelines. 25

Clin Chem. 57(6):919-21. 26

27

Halling KC, Schrijver I, Persons DL. (2012) 28

Test verification and validation for molecular diagnostic assays. 29

Arch Pathol Lab Med. 136(1):11-13. 30

31

Reising M, et al (2013) PCR Methods Comparison: a Guideline to Modify a Validated Assay. 32

Manuscript in preparation. 33

34

Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M. (2010) 35

A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines. 36

Methods 50(4):S1-5. 37

38

39

6

Determination of Ct cutoff values for negative samples 1 2 Establishment of Ct cutoff values for definition of negatives is an important consideration for the 3

PCR assay development process. Many labs set an arbitrary cutoff of 40 or 45 for defining 4

negative samples. Other labs use the linear range and limit of detection assay characteristics to 5

determine the cutoff. Current published recommendations suggest establishing a cutoff based on 6

the threshold values derived from lowest concentration of target sequence that can be detected 7

95% of the time. In practice, this often requires multiple repetitions at 2-fold dilutions around the 8

target endpoint and more time and money than the precision is worth. In either case, a cutoff 9

must be vetted by well characterized diagnostic samples (known positive and negative samples). 10

A suspect range is determined by analysis of these well characterized diagnostic samples. In 11

most labs 35-36 is the upper limit of positives; > 37 marks the start of weak positive or suspect 12

range. Confirmatory data should be obtained to support interpretation of CTs in the range of 37-13

40 or greater as positive. 14

15

Two examples of cutoff determination are described: 16

17

1) Method A: 18

19

Endpoint/cutoff values are determined for each assay after performing 12 replicates of the 20

standard curve using known copy number targets. The last 10 fold dilution at which there is 21

100% detection is used to establish a cutoff pos/suspect point at 2 standard deviations above the 22

average Ct at this dilution. An interpretive statement is included with suspect results. 23

A suspect result is likely due to detection of very low amounts of target sequence in the sample 24

and is not repeatable 100% of the time. Generally, this is followed up with repeated testing of 2 25

dilutions, including the endpoint concentration aliquoted into single use reactions in nucleic acid 26

dilution solution during further testing of the assay using clinical samples to more robustly test 27

the assay performance. 28

29

2) Method B uses the probit regression analysis as described in Burd et al, page 562. 30

31

32

33

Interpretation of the PCR result as well as the meaning of the Ct level depends upon the whole 34

picture. Pertinent factors/questions include: 35

is there clinical disease? 36 Is the agent endemic or an exotic agent? 37 Is it commonly found in animals or is it rarely diagnosed? 38 Is the agent ubiquitous in the environment? 39 Is the agent in a vaccine used on the premise? 40

41

42

7

Critical Issues for Conducting PCR Assays 1

The exquisite sensitivity of PCR assays requires the establishment of thorough and robust good 2

laboratory practice (glp). PCR-associated glp initiates with proper design of the physical space 3

and encompasses collection, transport and storage of samples; development and performance of 4

assays; use of proper controls and consistent interpretation of assays. Stringent quality 5

parameters are necessary to minimize contamination and to ensure consistent and reproducible 6

results across different reagent lots, labs, and personnel. The following recommendations are 7

provided primarily for BSL-2 laboratories; modifications for BSL-3 labs will be provided in 8

specific sections as needed. 9

10

I. Facilities: 11 The location of work areas should be established, as much as possible, according to the 12

appropriate building airflow direction. For example, the best scenario would be positive air 13

pressure in the Master Mix preparation room. 14

Note: positive air pressure is not an option for BSL-3 sections 15

16

Designate a minimum of 3 dedicated work stations: 17 18

I.1. Clean Reagent Preparation Area 19 This area is for preparation of nucleic acid purification reagents and PCR master mix 20

reagents only. No amplication targets (i.e. pathogens), plasmids, purified RNA or DNA, 21

and no amplified products should be allowed. Only trained personnel may use this 22

workstation. It is critical to have this area separated/dedicated as much as possible; at least 23

in a hood. Special attention to disinfection and cleaning of this area is essential. 24

I.2. Specimen Preparation Area 25 This area is for sample receiving and processing, and nucleic acid extraction and 26

purification only. 27

I.3. Target amplification/PCR Area 28 In this area the PCR instruments are located. 29

30

Ideally, work stations should be located in separate rooms. If needed (due to budget and space 31

constraints), however, work stations may be located in one room. Multiple hoods with 32

designated specific functions, i.e. master mix preparation, or nucleic acid purification reagent 33

preparation, may be placed in the same room. 34

35

Additional designated work stations may be established: 36

I.4. Addition of purified nucleic acids to the PCR master mixes. 37 Many laboratories use desk top PCR hoods. 38

39

I.5. Gel electrophoresis 40 Many laboratories have a designated room; other laboratories take extra precautions to 41

prevent aerosols such as freezing samples before opening tubes, and covering plates. 42

I.6. Preparation of amplification/extraction controls, standard curves, plasmids 43

8

There is strong agreement among laboratories that is very beneficial to have a separate area 1

for preparation of positive controls, particularly from material with a very high 2

pathogen/target concentration, i.e. cell culture supernatants, allantoic fluids, and high copy 3

number plasmid preparations, to avoid contaminating areas designated for diagnostic 4

samples processing. 5

I.7. nested PCR area 6 Some reference protocols still require the use of nested PCR. Some laboratories try to avoid 7

the risk of contamination by not offering this service, other laboratories offer the service 8

but are strictly designating the addition of the amplicon from the first step to the reagents 9

for the nested step to a hood. In case of space constraints, this step might be carried out in 10

the template-addition hood. 11

12

Additional measures may be employed to prevent contamination of the PCR workflow: 13

I.7.1. Establish a uni-directional physical workflow (pre-PCR to post-PCR) to avoid 14

contamination between work stations 15

I.7.2. Change PPE for each designated work area 16

17

II. Room environment and equipment 18

II.1. Temperature and humidity 19 Monitor temperature and humidity to enable proper functionality of instruments. Liquid 20

handling equipment, for example, is highly sensitive to changes in humidity. At this time, 21

reference values for acceptable ranges of temperature and humidity values have not been 22

established. 23

II.2. Monitor air and surface contamination in work stations 24 Establish schedules for monitoring: swab surfaces to check for surface contamination and 25

leave open tubes over night to check for aerosol contamination. A monthly schedule is 26

recommended. The PCR used may be the one that has the highest throughput in the lab. 27

Analyze data to determine source and frequency of contamination. 28

II.3. Work stations, hoods, biosafety cabinets 29 Hoods should be used for master mix preparation as much as possible. Desktop PCR 30

cabinets/workstations (with and without HEPA filters) provide enclosed and dedicated 31

spaces for different tasks when individual rooms are not available. The blower may be 32

turned on. Biosafety cabinets (BSL-2 hoods) may be used as well; they should be 33

certified annually. Hoods ducted to the outside may also be used for small amounts of 34

Trizol, Phenol, or beta-mercaptoethanol; check with applicable regulations. 35

II.4. Pipettors 36 Designate an individual set of pipettors for each work station. All pipettors should be 37

calibrated annually (certificate needs to be on hand). Depending on the frequency of use, 38

they should also undergo a functionality test (weight/volume check in the laboratory) at 39

least once a year or as often as necessary (i.e. after a malfunction that has been 40

corrected). Calibration may be performed by an external vendor. Functionality should be 41

verified by a designated (i.e. internally certified) technician upon return of the pipettor(s) 42

to the laboratory. 43

44

II.5. Thermocyclers: 45

II.5.1. Real-Time PCR Systems: 46

9

Read instrument manual for thorough understanding of system’s technology and 1

guidelines. Perform maintenance, background checks, and calibrations as 2

instructed by the manufacturer. For instruments under a manufacturer’s service 3

contract, occasional background checks should still be performed by the 4

laboratory. Monitor assay performance by use of appropriate controls for each 5

assay. Three consecutive runs resulting in assay failures for more than one assay 6

may indicate PCR system problems. 7

II.5.2. End-Point PCR Systems 8 Read instrument manual for thorough understanding of system’s technology and 9

guidelines. Monitor assay performance by using appropriate controls for each 10

assay. If desired or deemed necessary, perform system performance verification as 11

follows: 12

Perform serial (10x or 5x) dilution of an assay-specific amplification control. 13

Perform PCR on all dilutions and analyze amplicons using an appropriate 14

percentage agarose gel. Determine the highest dilution factor that produced 15

amplicons which are still visible on the agarose gel. Use a dilution which is 10-16

100 times more concentrated than the weakest signal in order to test each well in a 17

thermocycler. Perform the PCR, and analyze amplicons on agarose gel. When all 18

wells of the plate have produced a band of about equal intensity, the instrument 19

can be considered fully functional. 20

II.6. Ice buckets 21 There are multiple options; one or more can be selected as necessary and applicable for 22

each laboratory. 23

II.6.1. Disposable Styrofoam containers (soup cup size) 24

II.6.2. Designated ice beads containers/ice buckets for each work station 25

II.6.3. Clean Styrofoam shipping boxes that may be discarded weekly 26

II.7. Automated Extraction Instruments 27 Perform maintenance, background checks, and calibrations as instructed by the 28

manufacturer and following AAVLD guidelines. If an internal control is used with each 29

extraction, monitor the performance of this control using the accepted ranges for the 30

internal control for verification of system performance. Also, if equipment is moved to 31

another location, or if it is otherwise deemed necessary then perform system performance 32

verification as follows: 33

Extract a complete plate of known positive samples in a matrix of choice to determine if 34

all the magnetic pins are performing properly. (The pins may become bent or misaligned 35

causing a failed extraction//purification) 36

37

III. Disinfection and cleaning 38 A stringent cleaning routine needs to be in place to keep all work areas, all bench top work 39

surfaces, and all hoods clean and disinfected. One or more of the following recommendations 40

may be selected as necessary. 41

III.1. Spray and wipe surfaces with 10% bleach followed by 70% alcohol to avoid corrosion 42

of metal 43

III.2. Spray and wipe surfaces using Virkon® S according to label instructions (except 44

California) 45

III.3. Wipe surfaces with bleach wipes (commercial or freshly made) or ‘bleach-in-a-bottle’ 46

10

III.4. Use the interior UV lights of hoods for their decontamination (timers turn automatically 1

off after 1-2 hours) whenever not in use and/or in between assay setups. Available 2

PRRSV PCR data indicate, however, that UV light alone is NOT a reliable way to 3

control contamination. 4

5

IV. Personnel: 6 Performing diagnostic PCR assays requires good laboratory techniques and highly trained 7

personnel to ensure validity of results. Recommended requirements for new personnel are 8

B.S. degree or equivalent training in Biology, Microbiology, Virology, Biotechnology or 9

Bioengineering, Biochemistry/Chemistry, and equivalent. Preferred skills include analytical, 10

mathematical, organizational, and communication skills. Preferred attributes include 11

dedicated, self-motivated; shows attention to detail, good work ethics, team-oriented person. 12

Preferred experience includes repetitive tasks, and handling and processing diverse biological 13

and environmental samples. 14

Personnel of a PCR laboratory should be cross trained on all methods and processes to ensure 15

uninterrupted workflow. Cross training and rotations may also provide better job satisfaction. 16

The laboratory should have a proficiency test plan in place that all relevant personnel should 17

participate in. 18

19

V. Reagents Quality Control: 20 Core reagents and supplies should be purchased from reputable vendors with good quality 21

control practices to ensure best results and cost efficiencies. In-house prepared reagents are 22

acceptable with sufficient quality control testing. If laboratories use bulk mastermixes (all 23

components except target), laboratories must have data to demonstrate that there is no 24

significant decrease in assay sensitivity (e.g. positive and negative PCR control, trend tracking 25

or refer to Method Comparison document). 26

Perform Quality Control testing on each batch of in-house reagents. Quality Control testing 27

should include comparison of “test” and “control” lots of reagents; “test” denotes newly 28

prepared lot and “control” denotes previously tested lots. Passing criteria should be 29

established for specific reagents. Re-quantification of primers and probe upon receipt from the 30

manufacturer is considered a best practice in some laboratories. A method for primer/probe 31

QC is detailed at the Life Technologies website. Include appropriate extraction and 32

amplification controls for each PCR test to monitor consistent and acceptable assay 33

performance (refer to table in Section VII). 34

35

VI. Sample Acquisition and Handling 36

37

VI.1. Sample Acquisition 38 Proper collection of samples by technicians and veterinarians is important in order to obtain 39

PCR results that are interpretable. Laboratories should post collection instructions on their 40

web site. The instructions should begin with sample acquisition on farm and in the clinic. 41

Volume of sample and tube size (automation) should be specified. Recent vaccine history 42

including type of vaccine and route should be recorded because modified live vaccine 43

agents can be detected for weeks post vaccination. Vaccination at the same time of sample 44

collection should be discouraged due to contamination concerns. Practitioners must also be 45

cognizant of potential contamination from other environmental sources such as clothing 46

11

and gloves. Blood samples should be collected using a new syringe and needle for each 1

animal. Equipment should be disinfected with hypochlorite or peroxide based solutions 2

because these degrade nucleic acid. However, thorough rinsing is required to prevent false 3

PCR negative results due to residual bleach or peroxide. The timing of sample collection is 4

critical. Viral infections are usually best detected when sampling occurs within 48 hours 5

after onset of clinical signs. Consult laboratory if collection is outside of that time window. 6

Alternatively, shedding of virus or bacteria may be intermittent and require collection on 7

consecutive days. Instructions for pooling should be clearly stated as to whether this can 8

occur on farm or will be done in the laboratory. Validation data should support pooling of 2 9

or more samples. Often, pooling potential is predicted from the CT level of individual 10

samples and is not empirically derived. Clients should be informed whether it is a predicted 11

number or derived from actual testing. Some veterinary practices require use of substances 12

that may contain PCR inhibitors. Among these are: tattoo ink, milk containers for earnotch 13

samples, EDTA and heparin anticoagulants, bleach, and bacterial transport media. Proper 14

controls to test for the presence of inhibitors are recommended (section VII). 15

16

VI.2. Sample transport 17 Optimally, samples suspected of containing viral, mycoplasma or chlamydial agents are 18

best obtained with dacron or polyester tip, plastic applicator shaft, and sent chilled in viral 19

transport media. Alternatively, a sealed, sterile vial with several drops of saline can be a 20

satisfactory alternative for viral and bacterial detection. Even dry swabs can be suitable if 21

validation data exists to support their use. The following have been found to be unsuitable: 22

viral transport media brands which are stored at room temperature, calcium alginate swabs 23

and bacterial transport media. Samples should be sent using cold packs and next day 24

delivery recommended. Some sample types require special conditions, e.g. semen should 25

be shipped in a liquid nitrogen vapor tank. 26

27

VI.3. Processing at the Diagnostic lab 28 In the laboratory, decontamination of instruments used for preparation of samples should 29

be described in laboratory protocols or disposable tools should be used (e.g. scalpel, biopsy 30

punches or microtome blades). Provide instruction regarding decontamination of surfaces 31

and instruments used in necropsy. Samples potentially containing viral agents should be 32

refrigerated and tested within 24 hr or stored at -70/-80C. Samples potentially containing 33

certain bacteria or parasites (e.g. Cryptococcus) should not be frozen because pre-34

enrichment may be necessary prior to PCR amplification. Same quality and suitability 35

should be carefully assessed prior to processing. Unsuitable swabs, transport media, or 36

sample type as well as degradation may require rejection or a qualifying comment. In some 37

cases an inappropriate sample is the only sample available. The client should be informed 38

of the unsuitable sample and if the sample is still tested, results should be reported with a 39

cautionary statement. 40

41

42

43 44 45 46 47

12

VII Appropriate controls- The recommended controls are described in this chart 1

Controls Definition

What is it?

Purpose Recommended Best

practice

Extraction/purification controls

Internal

control

1. Amplifiable synthetic

nucleic acid (e.g. ultramer,

xeno RNA) added to every

sample or endogenous

housekeeping gene (e.g.18s).

2. Present in all samples tested

*

Assess efficacy of

the extraction and

purification method

for each sample.

Also assess presence

of inhibitors which

may vary from

sample to sample

No, with the following

qualifier:

After a date to be

determined it is strongly

recommended that new or

replacement assays

incorporate an internal

control.

Yes

Positive for the

extraction/purif

ication step(s)

control (one

per run/plate)

1. A well characterized

positive field sample in a

relevant matrix; or synthetic

nucleic acid containing the

target sequence spiked into the

lysis solution

2. Present in one well of each

assay

Assess efficacy of

reagent batch and

technical

performance. Also

assess reagent lots

and technical

performance

reproducibility

A positive extraction

control for each matrix

type is recommended

PEC not necessary if IC is

used. Yes

Negative

extraction /

purification

control (one

per run/plate)

1.Buffer , PBS, DMEM, VTM,

BHI (if the kit contains

sufficient carrier) or

well characterized negative

field sample. 2. Present in one

well of each assay

Assess target

contamination in

extraction reagents Yes Yes

Amplification controls

Positive

amplification

Control

1. Synthetic nucleic acid or

purified target from a well

characterized field sample or

reference material.

2. Present in one well of each

assay/run

Assess PCR

mastermix

functionality

Yes

Required even if positive

extraction control is used

Yes

No template

control (NTC)

All the components of the PCR

master mix with sterile nuclease

free water in place of template

2. Present in one well of each

assay

Assess target

contamination in

PCR reagents

Yes Yes

qPCR only

Copy number

control

1. Transcribed RNA or

synthetic DNA that can be

quantified without associated

cellular nucleic acid

2 Must generate a standard

curve

Control to monitor

the LOD or copy

number detection

Yes if performing

qPCR

No if performing relative

rPCR as is done in most

Vet-D labs

Yes if

performing

qPCR

13

VII. References 1

2

Sample Transport and Handling 3 1. Cloud JL, Hymas W, Carroll KC (2002) 4

Impact of nasopharyngeal swab types on detection of Bordetella pertussis by PCR and culture. 5

J Clin Microbiol 40:3838-40. 6

7

2. Daum LT, Worthy SA, Yim KC, Nogueras M, Schuman RF, Choi YW, Fischer GW (2011) 8

A clinical specimen collection and transport medium for molecular diagnostic and genomic 9

applications. 10

Epidemiol Infect 139:1764-73. 11

12

3. Druce J, Garcia K, Tran T, Papadakis G, Birch C (2012) 13

Evaluation of swabs, transport media, and specimen transport conditions for optimal detection of 14

viruses by PCR. 15

J Clin Microbiol 50:1064-5. 16

17

4. Gibb AP, Wong S (1998) 18

Inhibition of PCR by agar from bacteriological transport media. 19

J Clin Microbiol 36:275-6. 20

21

5. Payungporm S, Crawford PC, Kouo TS, Chen LM, Pompey J, Castleman WL, Dubovi EJ, 22

Katz JM, and Donis RO (2008) 23

Influenza A virus (H3N8) in dogs with respiratory disease, Florida. 24

Emerg Infect Dis 14:902-908 25

26

Internal Control 27 6. Hoorfar J, Malorny B, Abdulmawjood A, Cook N, Wagner M, Fach P (2004) 28

Practical considerations in design of internal amplification controls for diagnostic PCR assays. 29

J Clin Microbiol 42:1863-8. 30

31

7. Radonić A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A (2004) 32

Guideline to reference gene selection for quantitative real-time PCR. 33

Biochem Biophys Res Commun 313:856-62. 34

35

8. Radonić A, Thulke S, Bae HG, Müller MA, Siegert W, Nitsche A (2005) 36

Reference gene selection for quantitative real-time PCR analysis in virus infected cells: SARS 37

corona virus, Yellow fever virus, Human Herpesvirus-6, Camelpox virus and Cytomegalovirus 38

infections. 39

Virol J 2:7 40

9. Schroeder ME, Bounpheng MA, Rodgers S, Baker RJ, Black W, Naikare H, Velayudhan B, 41

Sneed L, Szonyi B, Clavijo A (2012) 42

Development and performance evaluation of calf diarrhea pathogen nucleic acid purification and 43

detection workflow. J Vet Diagn Invest 24:945-53. 44

45

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Glossary of Terms – Adapted from the OIE Quality Standard 1

2 1. Accuracy - nearness of a measurement to a known/actual/true value. A reference standard of a known 3 value can be used to assess the accuracy of a test method. 4 5 2. Analyte / Target - analyte and target are used interchangeably in polymerase chain reaction (PCR) 6 methods. A specific component of a test sample that is detected or measured by the test method. 7 8 3. Control sample -Sample(s) or reagents incorporated in every run of the assay to determine whether the 9 assay is performing within predetermined statistical parameters. 10 Types of controls include: 11

Extraction control (positive and negative) – to assess the efficiency of extraction process 12 Internal control (could be synthetic nonsense sequence) test for inhibitors and efficiency of 13 extraction 14 Amplification control (positive and negative) – to assess the PCR reaction efficiency 15 No template control (NTC) – to evaluate for reagent contamination 16

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4. Cut-off - the value used to distinguish between negative and positive results; may include 18 indeterminate or suspicious zone. 19 20 5. Cycle threshold (Ct) – the cycle at which a value rises above the threshold. 21

22 Threshold* – A level of delta Rn used for the Ct determination in real-time assays. The level is 23 set to be above the baseline and sufficiently low to be within the exponential growth region of the 24 amplification curve. The threshold is the line whose intersection with the amplification plot 25 defines the Ct (threshold cycle). 26

27 6. False-negative reaction - a known positive sample testing negative. 28 29 7. False-positive reaction - a known negative sample testing positive. 30 31 8. Fitness for purpose - characteristics of a test method and related procedures that relate to their 32 suitability for one or more specific diagnostic application(s). 33 34 9. Linearity - defines the capacity of the method to obtain test results proportional to the concentration of 35 the target analyte. 36 37 10. Linear range - interval of analyte concentrations over which the method provides suitable precision 38 and accuracy. 39 40 11. Matrix - all the constituents of a test sample with the exception of the analyte. 41 42 12. Optimisation - process by which all of the physical, chemical and biological parameters of an assay 43 are evaluated and adjusted in order to ensure that the performance characteristics of the test method are 44 best suited to the intended application. 45 46 13. Performance characteristic - an attribute of a test method that may include analytical sensitivity and 47 specificity, accuracy and precision, diagnostic sensitivity (DSe) and specificity (DSp) and/or repeatability 48 and reproducibility. 49

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1 14. Precision - the degree of dispersion of results for a repeatedly tested sample tested under similar 2 conditions. 3 4 15. Predictive Value (Negative) - the probability that an animal is free from exposure or infection given 5 that it tests negative; predictive values are a function of the Diagnostic sensitivity (DSe) and Diagnostic 6 Specificity (DSp) of the assay and the prevalence of infection. 7 8 16. Predictive Value (Positive) - the probability that an animal has been exposed or infected given that it 9 tests positive; predictive values are a function of the DSe and DSp of the assay and the prevalence of 10 infection. 11 12 17. Prevalence - the proportion of infected animals in a population at one given point in time. 13 14 18. Repeatability - level of agreement between replicates of a sample both within (intra-assay) and 15 between (inter-assay) runs of the same test method in a given laboratory. 16 17 19. Reproducibility - ability of a test method to provide consistent results when applied to aliquots of the 18 same sample tested by the same method in different laboratories. 19 20 20. Robustness - measure of an assay’s capacity to remain unaffected by small changes or variations 21 introduced in test conditions to mimic anticipated routine laboratory operation, part of optimization 22 studies and reflected in repeatability assessments (e.g. incubation times, reaction temperatures, buffer 23 pH/ionic strength, reagent dilutions, sample condition and/or preparation, etc.). 24 25 21. Ruggedness - a measure of an assay’s capacity to remain unaffected by substantial changes or 26 substitutions in test conditions anticipated in multi-laboratory utilization, part of fitness studies and 27 reproducibility assessments (e.g. shipping conditions, technology transfer, reagent batches, equipment, 28 testing platforms and/or environments). 29 30 22. Sensitivity 31

a. Analytical Sensitivity - synonymous with ‘Limit of Detection’, smallest detectable amount of 32 analyte that can be measured with a defined certainty. 33

34 b. Diagnostic Sensitivity (DSe)- proportion of known infected reference animals that test 35 positive in the assay; infected animals that test negative are considered to have false-negative 36 results. 37

38 c. Relative Sensitivity - proportion of reference samples, defined as positive by one or a 39 combination of test methods that also test positive in the assay being compared. 40 41

23. Specificity 42 a. Analytical Specificity - degree to which the assay distinguishes between the target analyte and 43 other components in the sample matrix. 44

45 b. Diagnostic Specificity (DSp) - proportion of known uninfected reference animals that test 46 negative in the assay; uninfected reference animals that test positive are considered to have false-47 positive results. 48

49 c. Relative Specificity - proportion of reference samples, defined as negative by one or a 50 combination of test methods, that also test negative in the assay being compared. 51

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1 24. Standardization - process by which a test method is calibrated to a uniform level of analytical 2 sensitivity and adjusted to consistent level of diagnostic agreement when compared to a recognized 3 standard test method, usually achieved by use of calibration reagents and test panels that are typically 4 provided by a reference laboratory. 5 6 25. Validation - process through which a test method is confirmed to be fit for its intended purpose, 7 against specified requirements for a particular diagnostic application; may also include continuous 8 monitoring. 9 10

a. Bench/Analytical Validation – process through which the analytical performance 11 characteristics of a test method are optimized and standardized, may include analytical sensitivity 12 and specificity, accuracy and precision, repeatability, and robustness criteria, as well as, 13 analytical comparisons to other test methods. Note - this process necessarily precedes Field 14 Validation, and an assay cannot be considered fit for most diagnostic purposes without the latter. 15

16 b. Field/ Diagnostic Validation - process through which the performance characteristics (may 17 include diagnostic sensitivity and specificity) of a test method are established using diagnostic 18 samples, field samples or samples from experimentally infected animals. Note - recommended 19 only for assays that have been thoroughly bench validated. 20 21 c. Verification - process by which a laboratory demonstrates a previously validated assay will 22 perform according to its established parameters. 23

24 Types of PCR: 25 Conventional PCR – real time PCR (rPCR)– quantitative real time PCR 26 The PCR method originally invented by Kari Mullis is still common to the PCR methods used today. The 27 basic principle is still the exponential amplification of sometimes minute amounts of target nucleic acids 28 in a sample. The difference between the methods lies in the data analysis. 29 30 Conventional PCR 31 Conventional PCR requires only a standard thermocycler with heating and cooling capability and is also 32 called end-point PCR: once DNA amplification has been terminated (typically after 40 to 45 cycles) an 33 aliquot of the resulting DNA has to be run on an agarose gel to visualize whether the PCR has been 34 successful, i.e. if there is an amplicon present. Determinations of DNA concentration can be 35 accomplished by running a molecular weight and concentration ladder on the gel with the sample but 36 quantitation in general is not very accurate and does not allow inferences to be made about the 37 concentration of target in the original sample. The limit of detection of DNA in an agarose gel is about 5-38 10ng so it is possible that a weak positive result goes unnoticed. 39 40 Real time PCR (TaqMan assay, 5’ nuclease assay) 41 This method employs, in addition to the primers used in conventional PCR, a third oligonucleotide, 42 commonly called probe. Real time PCR takes advantage of the 5’ to 3’ exonuclease activity of the Taq 43 polymerase. The probe must be complementary to the target DNA located between the two primers and 44 the melting temperature must be several degrees higher than the primers’ melting temperature so that it 45 binds to the target before the primers do. The probe is labeled at the 5’ end with a fluorescent dye 46 (reporter dye) and at the 3’ end with a quencher. The immediate proximity of the reporter and the 47 quencher prevents fluorescence but as soon as the primers have annealed, Taq polymerase has bound and 48 replication commences, the probe is degraded through the Taq’s exonuclease activity. Once removed 49 from the quencher’s immediate proximity, the reporter dye starts fluorescing when externally excited and 50 the increase of fluorescence over time indicates the PCR reaction is successfully carried out. 51

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Real time PCR instruments incorporate visualization of the PCR results as the software measures 1 any changes in fluorescence over the time of the cycling and computes them in relation to a reference dye 2 that is added to the reaction and does not change. The resulting graph in the case of a positive reaction 3 shows the lag time (amplification below the threshold), the exponential phase (amount of fluorescence is 4 proportional to the amount of target in the sample), and the plateau phase (reagents start to exhaust, no 5 more amplification is taking place). Thresholds and baseline values need to be set to enable data analysis. 6 While real time instruments are much more expensive than conventional cyclers and the TaqMan probes 7 are very expensive, too, the fact that no agarose gel is required for visualization of results and data 8 analysis represents a significant advantage. The relationship between visible fluorescence and amount of 9 target DNA in the sample makes the standard real time PCR semiquantitative. 10 MGB (minor groove binder) and LNA (locked nucleic acid) probes represent modifications of the 11 standard TaqMan probe that allow for a shorter probe with no tolerance for mismatches between probe 12 and target. DNA intercalating dyes like SYBR Green can be added to a real time PCR reaction in place of 13 a probe as it only fluoresces in the presence of double-stranded DNA, i.e. when PCR has been successful 14 and an amplicon has been created. 15 16 Semiquantitative real time PCR [relative quantification by real time (rPCR) or reverse 17 transcriptase real time PCR (rRTPCR) 18 The absolute quantitation of nucleic acid targets requires extensive and very costly extra effort (standard 19 curve, running in triplicates) and, therefore, those assays are rarely performed in veterinary diagnostic 20 settings. Alternatively, a relative quantification can be performed. Here, the CT of a sample is used by 21 itself - in relation to a thoroughly characterized positive control for the run and an equally well 22 characterized internal control in every well – for relative or rough estimation of amount of target nucleic 23 acid in the sample. Highly consistent performance of the positive control and the internal control are 24 required for those estimates to be meaningful. 25 26 Quantitative real time PCR 27 Due to the linear range of amplification when the amount of fluorescence is directly proportional to the 28 amount of target, the real time PCR offers the possibility to actually quantitate the amount of DNA or 29 RNA in a sample. While the actual method is essentially the same, quantitation requires the addition of 30 standard samples of known quantity to be run with the unknown samples. A typical standard curve 31 consists of a serial 10-fold dilutions of a sample where DNA concentration has been determined and is 32 expressed as DNA copies/ml or /ul. The more points the standard curve has the better it can perform. 33 Ideally, the linear range encompasses up to 7 points, i.e. from 10

7 to 10

1 copies/ml. Standard curve points 34

and the samples should be run in triplicate to be statistically meaningful and to enable calculation of 35 uncertainty of measurement. The known quantities need to be entered into the software and once the run 36 has finished, the software compares the Ct of the standard curve points with the Ct of the samples and 37 estimates the quantity. Strict quality parameters should be employed, (i.e. slope and r

2), to make the 38

quantitation useful. 39 40 Acronyms 41 1. Real-Time Polymerase Chain Reaction (DNA template) rPCR 42 2. Real-Time Reverse Transcriptase Polymerase Chain Reaction (RNA template) RT-rPCR 43

44 OIE is the source document for definitions #1-25 in this document 45 * definition used with permission from Applied Biosystems for Life Technology 46

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