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Implementation and validation of avian influenza virus TaqMan ...

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Australian Biosecurity Cooperative Research Centre for Emerging Infectious Disease

Project AL.037R

AB-CRC TECHNOLOGY TRANSFER SUPPORT

(APPLICATION & LINKAGE PROGRAM)

Report

Implementation and validation of avian influenza virus TaqMan

assays on various platforms in different state veterinary

diagnostic laboratories

Hans Heine and Lee Trinidad

CSIRO Livestock Industries

Australian Animal Health Laboratory

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TaqMan assays Technical report

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Participating laboratories

Australian Animal Health Laboratory (AAHL)

CSIRO Livestock Industries, Geelong Vic

Biosecurity Sciences Laboratory

Department of Primary Industries and Fisheries, Yeerongpilly QLD

Elizabeth Macarthur Agricultural Institute (EMAI)

NSW Department of Primary Industries, Menangle NSW

Primary Industries Research Victoria (PIRVic)

Department of Primary Industries, Attwood Vic

Animal Health Laboratories

Agriculture Western Australia, South Perth WA

Mt Pleasant Animal Health Laboratories – Diagnostic Services

Department of Primary Industries, Water & Environment, Launceston Tas

Department of Primary Industries and Resources, South Australia (*)

Gribbles Pathology, Wayville SA

(*) Operation has been transferred to Gribbles in Victoria

Investigation and Diagnostic Centre

Ministry of Agriculture & Forestry, Upper Hutt, New Zealand

Berrimah Veterinary Laboratories (**)

Department of Primary Industry, Fisheries and Mines; Berrimah, Northern Territory

(**) laboratory joint program later in 2006

Acknowledgements

Many thanks to all participants from the state veterinary diagnostic laboratories for their

cooperation and valuable discussions. Thanks to the members of the Molecular Diagnosis and

DSR group at AAHL or their help in test performance, evaluation and technology transfer. Thanks

to Dr Debby Cousins, AB-CRC & SCAHLS for facilitating this project.

This project was supported by CSIRO Livestock Industries and received funding as AB-CRC

TECHNOLOGY TRANSFER SUPPORT, APPLICATION & LINKAGE PROGRAM, Project

AL.037R from AB-CRC for Emerging Infectious Disease, RIRDC chicken meat program and

Australian Egg Corporation Limited (AECL).

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Contents

Participating laboratories ................................................................................................................. 2

Acknowledgements.......................................................................................................................... 2

Contents .......................................................................................................................................... 3

Summary ......................................................................................................................................... 4

Aims............................................................................................................................................. 4

Background ................................................................................................................................. 4

Methodology................................................................................................................................ 4

Outcomes and Benefits............................................................................................................... 4

Introduction ...................................................................................................................................... 5

Methodology .................................................................................................................................... 9

Strategy ....................................................................................................................................... 9

Kit configuration (Panels) ............................................................................................................ 9

Kit No. 1 .................................................................................................................................. 9

Kit No. 2 ................................................................................................................................ 10

Preparation of samples for kits.................................................................................................. 10

Shipment and storage of samples............................................................................................. 10

Evaluation and reporting ........................................................................................................... 10

Results and discussion.................................................................................................................. 10

Summary Laboratory Analysis .................................................................................................. 10

Examples of experimental results ............................................................................................. 14

Implications.................................................................................................................................... 18

Recommendations......................................................................................................................... 19

References .................................................................................................................................... 20

Appendix 1: Results Questionnaire sent to each laboratory ........................................................ 21

Appendix 2: Follow up from SCAHLS AI workshop July 2006 ..................................................... 22

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Summary

Aims

• To transfer avian influenza (AI) TaqMan RT-PCR assays from AAHL to state veterinary

diagnostic laboratories.

• To support the implementation of AI TaqMan RT-PCR assays in state veterinary

diagnostic laboratories.

• To evaluate the performance of the AI TaqMan RT-PCR tests on the different instruments

and various reagents routinely used by the respective laboratories.

Background

In response to the wide spread of highly pathogenic AI H5N1 in Asia in early 2004, realtime

RT-PCR diagnostic tests for rapid and sensitive detection of AI H5N1 were

developed at AAHL with support from the Australian Biosecurity CRC for Emerging

Diseases (AB-CRC project 1.001R), the Australian government Rural Industries

Research and Development Corporation (RIRDC) Chicken Meat Program, and the

Australian Egg Corporation Limited (AECL) (RIRDC project No CSA-24J). The project

(AB-CRC project AL.037R) described in this report was established to transfer the new

tests to state veterinary diagnostic laboratories and to evaluate the performance of the

tests on the various instrument platforms used in the respective laboratories.

Methodology

• AAHL provided protocols, technical information and supplied positive and negative

controls and samples of inactivated AI virus to the participating state laboratories.

• Each participating laboratory implemented and evaluated the tests on their existing

instrument and reported test results to AAHL. Test results were analysed and critical

parameters from different platforms were identified in consultation with the participating

laboratories.

• Standard operating procedures and test validation data from AAHL will be submitted to

SCAHLS.

Outcomes and Benefits

• All participating laboratories have set up the Influenza Type A and the H5 TaqMan tests.

Increased capabilities in state veterinary diagnostic laboratories for rapid and sensitive

detection of H5N1 by real-time PCR will increase the chances of early detection in

diagnostic and surveillance samples.

• The identification and evaluation of critical technical parameters affecting test results by

different laboratories will increase confidence in the interpretation of diagnostic test

results in an outbreak and will facilitate the implementation of other real-time PCR assays

in future.

• A proficiency test program will be implemented for AI TaqMan RT-PCR.

• Implications and recommendations are provided with this report on page 18-19.

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Introduction

Highly pathogenic avian influenza (HPAI) is defined on the basis of in vivo and in vitro tests, and

on amino acid sequence of the cleavage site of the haemagglutinin. HPAI has been associated

only with subtypes H5 and H7, however, not all H5 and H7 viruses cause HPAI. Wild birds are

generally believed to be reservoirs for low virulence H5 and H7 viruses that can mutate to high

virulence after introduction into poultry (Alexander, 2000). Avian influenza has worldwide

distribution. Five outbreaks of HPAI have occurred in Australia, in 1976, 1985, 1992, 1994 and

1997. These outbreaks were all of the H7 subtype and are believed to have been associated with

migratory waterfowl. Many strains of non-pathogenic AI viruses have been reported in native and

migratory birds in Australia. Clinical signs in chickens range from sudden death to respiratory

distress, oedema of the head, cyanosis, and diarrhoea. Mortality rates vary from low to 100% for

HPAI, and are influenced by the host’s immune status, age, presence of other disease agents

and environmental conditions. Virus is shed in the faeces and respiratory tract and horizontal

transmission occurs readily. HPAI virus is sensitive to extremes of environmental conditions;

however its survival in organic matter and water forms an important source of virus in outbreaks

of disease in poultry. The HPAI H5N1 strains (Li et al., 2004) first detected in live bird markets in

Hong Kong in 1997 have spread throughout Asia into Europe and parts of Africa, infecting

domestic poultry and wild birds. H5N1 strains have also shown the potential for infecting other

species and have been associated with human fatalities, raising fears of a potential pandemic.

Many of the long-established laboratory diagnostic tests are time consuming, lack sensitivity or

cannot differentiate between closely related virus types. New real-time PCR diagnostic tests,

based on the intrinsic specificity of probes such as TaqMan probes (Holland et al., 1991), are fast

and highly sensitive, enabling the differentiation of closely related isolates in a few hours without

the need for agarose gel electrophoresis and DNA sequencing. These tests can be automated for

screening large numbers of samples for different viruses to support epidemiological surveys

following an outbreak. TaqMan RT-PCR assays for detection of all Type A influenza strains and

the H5 haemagglutinin gene (HA) of the Eurasian lineage of influenza have been developed at

AAHL (Fig.1). Both assays utilise the highly conserved gene-specific fluorescent probes

developed for American isolates (Spackman et al., 2002) and newly designed forward and

reverse primers incorporating multiple nucleotides homologous to sequences of Eurasian H5N1

isolates (Heine et al., 2005; Heine et al., 2007). A new TaqMan assay specific for Australian H7

strains was developed (Heine and Trinidad, 2006) as these strains form a distinct subgroup within

the Eurasian lineage (Banks et al. 2000) which was also confirmed by the analysis of the newly

sequenced (GenBank AY943924) HPAI isolate from the 1997 outbreak in NSW. The tests have a

wide linear range of detection over 5 to 6 logs of template concentration and are suitable for

detection and differentiation of H7 and H5 strains in various clinical samples. Such a high

analytical sensitivity makes the tests valuable for detection of virus in surveillance studies where

only low viral loads are expected in infected birds. The purpose of AI TaqMan tests is the

detection of a broad range of AI strains and the differentiation of H5 (H5N1) strains to investigate

clinical signs in index case diagnosis and for AI exclusion in samples from wild bird species in

surveillance.

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Target genes for AI TaqMan assays

Seg

1

2

3

4

5

6

7

8

Protein

PB2

PB1

PA

HA

NP

NA

MA

NS

Heine AAVLD 20051026

Function

Polymerase component

Polymerase component

Polymerase component

Hemagglutinin > H5 & H7 specific

Nucleocapsid

Neuraminidase

Matrix protein (M1 + M2) > type A

Nonstructural protein (NS1 + NS2)

Figure 1. Genome targets for AI TaqMan assays.

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TaqMan

Assays

(One-step)

Technology and factors influencing TaqMan real-time PCR.

The TaqMan real-time PCR technology and various factors affecting the performance of such

assays have been widely described (Holland et al., 1991; Bustin and Nolan, 2004; Wacker and

Godard, 2005; Reynisson et al., 2006; Ambion TechNotes, 2007). Main advantages of the

technology are its high sensitivity and specificity, the simplicity and speed of the assay, its

suitability for automation and the reduced risk for cross contamination. Internal controls for house

keeping genes, 18S rRNA or others can be included utilizing multiplexing capabilities of most

instruments. Quality of the nucleic acid template, especially RNA is a major factor for test

performance and requires special care to avoid co-purification of RT-PCR inhibitors and

nucleases (RNase activity) that lead to failure of the reaction resulting in false negative results.

Thermo-stable enzymes contained in the kits of different manufacturers are derived from various

sources and their properties affect reaction efficiencies. Other factors and the combination of

fluorescent dyes as well as the inclusion of a reference dye such as ROX affects the readout of

the reaction. Thermocycler instruments are built to a number of design principles affecting

temperature cycling and the optical systems to excite and measure the fluorescent signal.

Essential for the control of cross-contamination are the implementation of a QA system and

appropriate workflows.

Analysis and interpretation of TaqMan real-time PCR results.

The increase in reporter signal is captured by the instrument software and displayed as an

Amplification Plot when the threshold signal increases to a detectable level (Fig. 2). The

Threshold is the point of detection (above background). The Cycle–Threshold (Ct) cycle

(number) at which the sample crosses the threshold is the parameter most frequently and most

conveniently used to report RRT-PCR results. The Ct values can be misleading and insufficient to

draw conclusions without a thorough assessment of factors contributing to this measurement.

The threshold cycle (Ct) is defined as the cycle number when sample fluorescence exceeds a

chosen threshold above calculated background fluorescence. The background fluorescence is

influenced by changing reaction conditions and internal reference dyes such as ROX are used to

normalise variations in the baseline. The threshold calculated by the real-time instrument

depends on the baseline and the values of individual reactions within a single run of multiple

TaqMan assays Technical report

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eactions. The threshold must intersect the exponential phase of individual amplification plots and

may need adjustment within a range of values within the exponential phase of the amplification

plot. It is important to check instrument specific parameters and to set the baseline and threshold

properly.

Automated detection of PCR product increase during the amplification cycles

(1)

Heine AAVLD 20051026

(2)

Threshold cycle (C T ) when PCR product

crosses the threshold of detection

Figure 2. Amplification plot features. Modified from Applied Biosystems manual.

Threshold (1): The threshold is adjusted to a value above the background and significantly below

the plateau of an amplification plot. It must be placed within the linear region of the

amplification curve, which represents the detectable log-linear range of the PCR.

Threshold cycle (CT) (2) or crossing point: This is the cycle, at which the amplification plot

crosses the threshold, i.e., at which there is a significant detectable increase in

fluorescence. The CT serves as a tool for calculation of the starting template amount in

each sample.

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TaqMan amplification plots of 1/10 serial diluted RNA

Threshold

Heine AAVLD 20051026

Threshold

Cycle (CT)

neat

AI subtype H5 specific TaqMan assayt: Log 10 dilutions of viral RNA

A/chicken/Vietnam/39/2004 (H5N1) were tested in triplicates

Relative quantitation of template over ~10 6 -fold linear range

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RNA dilutions

Figure 3. Amplification plot of serial diluted RNA template.

most dilute (-6)

Test validation and technology transfer.

The basic assay parameters including optimization of primer and probe concentrations and the

determination of limits of detection and analytical sensitivities by testing serial dilution of positive

reference material were evaluated at AAHL on an Applied Biosystems 7700 sequence detection

system instrument prior to the technology transfer (Fig. 3) (see AB-CRC Technical Report). The

multiplexing capabilities of the instrument were utilized to include VIC labeled 18S-rRNA control

reactions in these tests. All tests were performed under the AAHL QA system for diagnostic

testing. At the outset of the technology transfer each of the veterinary diagnostic laboratories

participating in the technology transfer had already real-time PCR facilities established in their

laboratories. In total five different instrument platforms were evaluated in the nine participating

laboratories.

Variables between laboratories.

• Sample transport, RNA extraction and storage

o Each laboratory performed their RNA extractions from the provided samples.

• Primer / probe

o Each laboratory sourced their own primers and probes based on sequences from

AAHL.

• Different enzyme mixes

o Each laboratory used their preferred enzyme kits (Applied Biosystems, Invitrogen,

Roche; one-step, two-step reactions)

• PCR instruments

Differences in instruments can be thermocycler (rotor, heat block, ramping speed), optical

system (Laser, tungsten, LED, filters, photomultipliers), multiplex capability and software.

Some main differences of instruments were that Roche instrument required glass capillaries,

2-step reactions and dedicated enzymes. Applied Biosystems and BioRad instruments use

thermo block whereas Corbett and Roche do not. Real-time PCR instruments used were:

• AAHL: Applied Biosystems ABI 7700 and ABI 7500 Fast

• QLD DPI&F: Corbett Rotor-Gene RG3000

• NSW DPI (EMAI): ABI 7500 Fast (Cepheid Smartcycler; 2 nd instrument)

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• VIC PIRVIC: ABI 7500 (Roche LightCycler; 2 nd instrument)

• SA Gribbles: Corbett Rotor-Gene RG3000

• WA DPI: Biorad iCycler

• TAS DPIWE: Roche LightCycler

• NT DPIFM: Corbett Rotor-Gene RG6000

• NZ MAF: Corbett Rotor-Gene RG3000

Methodology

Strategy

• AAHL provided experimental protocols, sequences and technical advice for AI TaqMan

assays to all participating laboratories.

• AAHL prepared and distributed AI reagent kits (#1 and #2) containing inactivated virus

positive controls and coded unknowns.

• Each participating laboratory carried out primers and probe titration to determine the

optimum concentrations of use.

• Each participating laboratory set up the TaqMan assays on their respective instrument

platform following the SOP provided by AAHL as closely as feasible. Each laboratory

extracted viral RNA from the material provided in the kits and performed basic assay

evaluation to determine the limit of detection by serial dilution of the template. Further

optimization of primer and probe concentrations was carried out if necessary.

• Participating laboratories reported results back to AAHL for analysis and to identify if any

modifications of assay conditions were required for particular instrument platforms. AAHL

provided technical advice when requested and analysis of results has been discussed

with individual laboratories.

• Meeting of representatives from the different state veterinary laboratories was held at the

AI workshop at AAHL in July 2006.

• A report on the evaluation of data from the different laboratories, instruments and

reagents used, and recommendations the performance and implementation of the test

will be prepared and submitted to SCAHLS.

Kit configuration (Panels)

Kit No. 1

Kit #1 (multiple vials of strong positive AI from allantoic fluid inactivated in RLT extraction buffer

(Qiagen); negative controls from uninfected allantoic fluid)

5 x 750 µl aliquots of positive control sample [allantoic fluid infected with A/chicken/shearwater/75

(H5N3) in Qiagen RLT buffer + ß-mercaptoethanol]

2 x 750 µl aliquots of negative control sample [uninfected allantoic fluid in Qiagen RLT buffer + ßmercaptoethanol]

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Kit No. 2

Kit #2 (coded blind test samples including positive and negative)

Table 1. Identification code of samples in kit #2

Code Isolate

0602-07-1108 A/chicken/Cambodia/1A/2004 H5N1 (HP)

0602-07-1057 A/emu/NSW/1997 H7N4 (HP)

0602-07-1059 A/duck/Victoria/1976 H7N7 (LP)

0602-07-1100 A/grey teal/WA/1840/1979 H4N4 (LP)

0602-07-1001 A/chicken/Vietnam/8/2004 H5N1 (HP)

0602-07-1101 A/shelduck/WA/1762/1979 H15N9 (LP)

0510-11-0503 A/shearwater/Australia/1975 H5N3 (LP)

0602-28-0901 Uninfected* allantoic fluid – negative control

Preparation of samples for kits

Multiple vials containing AI infected or uninfected allantoic fluid inactivated in RLT extraction

buffer (RNeasy kit; Qiagen) ready for subsequent RNA extraction according to manufacturer’s

standard protocol were sent to participating laboratories. Inactivation of AI is based on the

concentration of guanidinium being greater than 20% in RLT lysis buffer (information supplied by

manufacturer). Each 750 µl sample aliquot contained 144 µl allantoic fluid inactivated in 600 µl

RLT extraction buffer (Qiagen) containing 6 µl ß-mercaptoethanol.

Shipment and storage of samples

Samples were shipped on dry ice. Upon receipt samples were recommended to be stored at -

80°C until needed. To maintain RNA integrity, we recommended extracting RNA just prior to

performing the assays. Small (single use) aliquots of the RNA may be stored at -80°C, but should

be used immediately after thawing and any remaining sample should be discarded.

Evaluation and reporting

Experimental results from different laboratories were collected at AAHL and analysed in

conjunction with the user. A report on the test performances and recommendations was provided

at a meeting of participants at AAHL in July 2006. A report will be submitted to SCAHLS and the

AB-CRC on the analysis of the validation trial data, comments and recommendations on the test

validation process, and identification of project-related issues or opportunities. A report on the

validation data and recommendations for SOPs for influenza Type A, subtype H5 and H7 specific

TaqMan assays and evaluation of different instruments and reagents will be prepared and

submitted to SCAHLS.

Results and discussion

Summary Laboratory Analysis

The AI samples in kit #1 served to provide positive and negative material for the initial setting up

of the TaqMan assays in each laboratory and facilitated the optimization of reaction conditions,

especially primer and probe concentrations and to determine the linear range of the assay by

serial dilution of template. The samples in kit #2 served to establish comparative data between

the different laboratories and to ensure that a range of different AI could be detected in each

laboratory. The laboratories were asked not to include the VIC-labeled 18S rRNA internal control

in the initial tests until the

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effects of an internal control were evaluated on instrument and enzyme combinations other than

the Applied Biosystems platform and suitability of different instruments for multiplexing was

established by the various laboratories.

The AI TaqMan results submitted from the different laboratories and technical issues with the

tests were discussed at a workshop for the National Standardisation of Avian Influenza

Diagnostic Tests held at CSIRO Australian Animal Health Laboratory in Geelong, 20 th – 21 st July

2006. The results of the TaqMan Type A assays expressed as Ct values for all samples from

kit#2 are shown in Figure 4. The Ct values are grouped on the Y-axis while laboratories are

arranged along the X-axis. Individual laboratories are not identified in this graph. The spread of Ct

values indicated that all laboratories were able to detect the relevant AI samples provided in the

kits. Ct values need to be interpreted very cautiously as they are not the sole determinants of

analytical sensitivity and they cannot be directly compared between laboratories as threshold

values used to determine the setting of Ct were not uniform across different laboratories.

The limit of detection of serial diluted positive control provided in the kits was log 5 in most

laboratories with a spread between log 4-6 dilutions in some laboratories. All instruments were

capable of detecting highly diluted AI genomic material. The type of thermocycler used was not

the major factor in variation between laboratories. Direct comparison of Ct values between

laboratories is not appropriate as the threshold value for setting Ct values was not uniform

between laboratories and a number of other factors can influence Ct values (Table 2). A high

enough concentration of PCR primers was critical especially for primers such as the reverse

primer in the type A assay that contained a higher degree of multiplicity by inclusion of redundant

nucleotides in various positions. The type of reaction enzyme mix also appeared to influence Ct

values and reaction efficiencies and this has been confirmed in subsequent experiments done at

AAHL and Biosecurity Sciences Laboratory (DPI&F Queensland). This may explain the

discrepancies observed in results obtained from some low positive field samples from

Queensland and Tasmania.

Cycle Threshold

35

30

25

20

15

10

5

0

AI Type A TaqMan results of kit #2 samples

0 2 4 6 8 10

Laboratory (instrument & assay platform)

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S1

S2

S3

S4

S5

S6

S7*

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Sample# Virus

S1 A/chicken/Cambodia/1A/2004 H5N1 (HP)

S2 A/emu/NSW/1997 H7N4 (HP)

S3 A/duck/Victoria/1976 H7N7 (LP)

S4 A/grey teal/WA/1840/1979 H4N4 (LP)

S5 A/chicken/Vietnam/8/2004 H5N1 (HP)

S6 A/shelduck/WA/1762/1979 H15N9 (LP)

S7 A/shearwater/Australia/1975 H5N3 (LP)

S8 Allantoic fluid (* uninfected, not show on graph)

Figure 4. Summary of AI type A TaqMan results of each laboratory from kit #2 samples

The main unresolved issues identified in the workshop were:

• To evaluate and implement the use of internal controls such as VIC labeled 18S rRNA

control reaction

• To establish a fixed threshold value by each laboratory to enable better comparison of

results between laboratories

• To determine limits of detection and set cut-offs for reporting TaqMan results

• To establish ways of interpreting and reporting indeterminate TaqMan results and

procedures for follow up action

Laboratories were advised on procedures to implement 18S rRNA internal controls, determine the

best threshold value, and determine cut-off values for positive, indeterminate and negative results

by using samples supplied in kit #2.

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The main differences in instrumentation, enzymes and reaction conditions used by the

laboratories for the testing of kit #2 samples are outlined in Table 2. A plus sign in square bracket

[+] indicates alternate options in the same laboratory.

Table 2. Differences between laboratories.

Laboratory

AAHL NSW Vic Ql

d

NZ SA NT WA Tas

RNA prep RNeasy + + + + + + + +

(Qiagen)

MagnaPure

total NA

(Roche)

[+]

Trizol [+]

High pure viral

RNA kit

(Roche)

+

Instrument ABI 7700 /

7500

+ + +

Corbett RG

3000

+ + +

Corbett RG

6000

+

BioRad iCycler +

RocheLightCy. +

Cepheid

Smartc.

[+]

Enzymes Applied Biosys.

(one-step) (*A)

+ + + +

Invitrogen Ss.

III (one-step)

(*B)

[+] + + + +

StrataScript RT;

Roche

TaqMan; (twostep)

+

Oligos [nM] 1200/1200/167 +

Fwd/Rev/Prob

e

900/900/250 + + + + +

800/800/200 +

400/800/100 +

600/800/200 +

Internal

18S

controls

rRNA

Data Analysis Threshold .05 .054 .01 .1 .01 .01 .0 200

value for Ct

(*C)

1

Interpretation Positive (*C) < 37 33 33 37 31 38 29 35

CT cut-off Negative (*C) > 40 38 38 40 37 42 33 38

Indeterminate 37-40

Different cut- Positive (*C)

36

40

off for H5 test Negative (*C)

40

45

(*A) TaqMan One-Step RT-PCR Master Mix (Applied Biosystems)

(*B) Superscript III Platinum one-Step Quantitative RT-PCR System (Invitrogen)

(*C) Threshold and Ct cut-off values used by laboratories for proficiency kit#3 results

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Further evaluation experiments were conducted by some of the laboratories after the workshop

and results were reported back to AAHL. Comparison of results between laboratories showed

that various threshold settings could lead up to 4 Ct value differences. The inclusion of ROX dye

for normalization seemed to affect the Ct values and systems with less or no ROX may have an

apparent lower Ct without changing the range of detection. Different filter systems in different

instruments will determine the capability for multiplexing especially near the lower limit of

detection.

Examples of experimental results

NOTE: In the following section results are presented from preliminary or single experiments to

illustrate a point and are not necessarily an indication of the current performance or capability of

particular laboratories.

Cycle threshold (CT)

Influence of threshold settings on CT value in two labs

Lab A: AI type A TaqMan results @ 3 thresholds

0

-6 -5 -4 -3 -2 -1 0

H5N1 RNA dilution

Heine AAVLD 20051026

45

40

35

30

25

20

15

10

5

Thr 0.01

Thr 0.05

Thr 0.1

Linear (Thr 0.05)

Cycle threshold (CT)

Lab N: AI type A TaqMan results @ 3 thresholds

45

40

35

30

25

20

15

10

5

0

-6 -5 -4 -3 -2 -1 0

H5N1 RNA dilution

Threshold settings had a bigger influence on CT value in lab N than in A

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Thr 0.01

Thr 0.05

Thr 0.1

Linear (Thr 0.05)

Figure 5. Influence of threshold settings on Ct values in two laboratories.

The threshold settings did influence the Ct values as expected, but could vary to different extend

between laboratories. The figure above shows that different threshold value settings (0.01, 0.05

and 0.1) had a greater influence in Laboratory N than A, that the difference was greater at higher

template dilutions in laboratory N than in laboratory A and that the reaction efficiencies as

measured by the slope of the graphs differed between laboratory A and N.

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Effects of different master mix and running mode

CT value

40

35

30

25

20

15

10

5

0

Heine AAVLD 20051026

Comparison Mastermixes and Running Modes

for AI Type A TaqMan

10-0 10-1 10-2 10-3 10-4

AI (H5N3) RNA dilution

Instrument mode: AB-7700, 7500 or 7500 FAST

Enzyme: AB or Invitrogen one-step kit

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AB-7700

AB-7500

Inv-7700

Inv-7500

Inv-Fast

Figure 6. Effects of different master mixes and running mode.

A simple comparison of effects of instrument and enzymes was done at AAHL. The old AB7700

instrument was compared with the new AB7500 in normal and Fast mode using either Applied

Biosystems or Invitrogen reagent kits. The instrument did not significantly change Ct values,

whereas greater differences were observed between AB and Invitrogen enzyme kits. Although

the Invitrogen enzyme kit showed lower Ct values than AB, this could not be interpreted as higher

analytical sensitivity as both enzyme systems had the same linear range of detection.

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Difference in PCR efficiencies between two labs

Cycle threshold (CT)

Heine AAVLD 20051026

Comparison results lab A & N @ same threshold

y = -4.541x + 14.773

R 2 = 0.9965

y = -3.127x + 24.017

R 2 = 0.9922

0

-6 -4 -2 0

H5N1 RNA log dilution

45

40

35

30

25

20

15

10

5

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Thr 0.05 (A)

Thr 0.05 (N)

Linear (Thr 0.05 (N))

Linear (Thr 0.05 (A))

Figure 7. Differences in PCR efficiencies between two laboratories.

Different slopes of template dilution curves indicate significant differences in the PCR reaction

efficiencies between laboratory A and N.

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CT value

50

45

40

35

30

25

20

15

10

5

0

Example of reduced linear range of detection &

interference from control reaction

Type A TaqMan H5N1: Example +/- 18S control

Heine AAVLD 20051026

log RNA dil

Minus 18S

Plus 18S

RNA

log 0

log -1

log -2

log -3

log -4

log -5

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Minus 18S rRNA

Ave

29.6

28.5

31.8

35.9

40.1

43.6

STDV

0.3

0.1

0.2

0.1

0.3

1.3

Ave

32.4

31.8

35.7

40.7

Not linear;

short limit of detection;

interference from 18S rRNA control reaction

Plus 18S

rRNA

Figure 8. Examples of reduced linear range of detection and interference from control reaction.

The data from a single experiment in one laboratory indicated non-linearity of the assay at high

template concentrations with and without the 18S rRNA control and reduced sensitivity when 18S

rRNA control is included. This graph also demonstrates the merit of obtaining comprehensive

data from standard curves of serial dilutions and to be cautious in interpreting single point Ct

values.

STDV

0.4

0.6

0.5

0.9

TaqMan assays Technical report

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Wide linear range of detection – Example from one lab

CT value

45

40

35

30

25

20

15

10

5

0

Heine AAVLD 20051026

TYPE A Influenza Assay with H5N3 - QLD (with 20nm x 18S)

y = 3.6689x + 14.516

R 2 = 0.9995

y = 3.5921x + 13.284

R 2 = 0.9993

y = 3.4696x + 11.167

R 2 = 0.9992

Neat -1 -2 -3 -4 -5 -6

RNA Log Dilution

PCR efficiency ~90%

Page 18 of 23

Threshold = 0.01

Threshold = 0.05

Threshold = 0.1

Linear (Threshold = 0.01)

Linear (Threshold = 0.05)

Linear (Threshold = 0.1)

Figure 9. Wide linear range of detection – Example from one laboratory.

Example from one laboratory evaluating inclusion of 18S rRNA control reactions at different

settings.

Implications

1. The AI TaqMan RT-PCR assays have been set up on different platforms used in the

participating laboratories. All AI isolates in sample kits provided by AAHL were detected by

each participating laboratory. The implementation of the AI TaqMan assays in state

diagnostic laboratories will reduce the time for the identification of a potential AI emergency or

outbreak and will assist in the rapid activation of control measures to limit the spread of

disease.

2. The different types of instruments were not a major factor for variation of results between

laboratories, although different technical specifications in optical systems and software

appeared to affect the capabilities for multiplexing of FAM labeled test reactions with VIC

labeled internal control reactions. Problems with the implementation of internal control

reactions (VIC labeled 18S rRNA) are not yet resolved in a number of laboratories and

require further investigation.

3. Differences in the reaction conditions affected assay performance. Adequate primer and

probe concentrations for best analytical sensitivity have to be determined by checkerboard

titration, especially for oligonucleotides containing multiplicities (redundancies). The different

types of reaction enzyme kits (one or two step reactions, different manufacturer), employed

by various laboratories depending on their preference or recommendation by the instrument

manufacturer, did affect Ct values to a small degree. Test performance criteria should be

determined for each of the kits used in one laboratory.

4. The setting of uniform cut-off values across different platforms and laboratories is not

appropriate as the numerical Ct values are influenced by a number of factors that varied

TaqMan assays Technical report

Version 300407; Checked Hans Heine


etween laboratories. Each laboratory will have to establish their cut-off values based on

known reference samples. Laboratories have been provided with recommendations to

establish appropriate thresholds and determine cut-off values based on serial dilution of a

known positive controls.

5. Other issues such as laboratory proficiency, staff training, QA system, reproducibility etc were

not evaluated during the technology transfer and will need to be addressed in a future

national proficiency program.

Recommendations

1. All laboratories should continue to determine and monitor assay performance parameters on

their respective platforms using reference sample material provided by a central laboratory.

Each laboratory should establish appropriate thresholds and determine cut-off values and

limit of detection based on serial dilutions of standard positive controls. The laboratories

should continue to collaborate to monitor and improve tests and to adapt to technology

changes.

2. All laboratories should implement the use of internal controls (such as 18S rRNA) in a

multiplex reaction if possible. Laboratories have to ensure that the internal control reaction

does not interfere with the performance of the test reaction. Negative test results should be

regarded with caution in cases where internal controls have not been performed.

3. All positive test results should be interpreted in conjunction with other biological and clinical

findings as the highly sensitive PCR assays can detect traces of viral genome in samples

where virus cannot be isolated. There is a need for further investigation into the significance

of field samples that are real-time PCR positive but virus isolation negative.

4. Other aspects of the test that were not part of the technology transfer project should be

monitored where possible and results communicated between the laboratories to ensure that

the tests can be kept up to date. Some of the parameters to observe are the influence of

different bird species (chickens vs. ducks and wild birds); different sample types (cloacal and

tracheal swabs, tissues and allantoic fluid); different nucleic acid extraction procedures,

storage conditions of samples or extracted RNA. The assay performance should be validated

in the reference laboratory on any new isolate and available sequence data should be

monitored to identify genetic changes in circulating virus that may impact on test

performance.

5. A national proficiency testing program containing high and low positive, negative and

replicate samples should be implemented to monitor the performance of AI TaqMan RT-PCR

in all laboratories. Such a program would provide valuable comparative data on assay

repeatability, analytical specificity and sensitivity.

Page 19 of 23

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References

Alexander DJ (2000). A review of avian influenza in different bird species. Vet. Microbiol. 74, 3-13

Ambion TechNotes 10(2): Top ten most common real-time qRT-PCR pitfalls.

www.ambion.com/techlib/tn/102/17.html

Banks J, Speidel EC, McCauley JW, Alexander DJ (2000). Phylogenetic analysis of H7

haemagglutinin subtype influenza A viruses. Arch. Virol. 145, 1047–1058

Bustin SA, Nolan T (2004). Pitfalls of quantitative real-time reverse-transcription polymerase

chain reaction. Journal of Biomolecular Techniques 15: 155-166.

Heine HG, Trinidad L, Selleck P (2005). Influenza virus type A and subtype H5-specific real-time

reverse transcription (RRT)-PCR for detection of Asian H5N1 isolates. Technical Report

for Australian Biosecurity Cooperative Research Centre for Emerging Infectious Disease.

2005. Download from http://www1.abcrc.org.au/

Heine HG, Trinidad L (2006). Rapid identification and pathotyping of virulent IBDV, NDV and AIV

isolates. The development and implementation of laboratory tests for rapid detection and

differentiation of viruses. A report for the Rural Industries Research and Development

Corporation (RIRDC) Chicken and Meat Program. RIRDC Project No CSA-24J.

Heine HG, Trinidad L, Selleck P, Lowther S. (2007). Rapid Detection of Highly Pathogenic Avian

Influenza H5N1 Virus by TaqMan Reverse Transcriptase-Polymerase Chain Reaction.

Avian Diseases 51: 370-372.

Holland PM, Abramson RD, Watson R, Gelfand DH (1991). Detection of specific polymerase

chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus

DNA polymerase. Proceedings of the National Academy of Sciences of the United States

of America 88, 7276-7280.

Li KS, Guan Y, Wang J, Smith GJ, Xu KM, Duan L, Rahardjo AP, Puthavathana P, Buranathai C,

Nguyen TD, Estoepangestie AT, Chaisingh A, Auewarakul P, Long HT, Hanh NT, Webby

RJ, Poon LL, Chen H, Shortridge KF, Yuen KY, Webster RG, Peiris JS (2004). Genesis

of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia.

Nature 430, 209-213. 2004.

Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, Perdue ML, Lohman K, Daum LT,

Suarez DL (2002). Development of a real-time reverse transcriptase PCR assay for type

A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J. Clin. Microbiol. 40,

3256-3260. 2002.

Reynisson E, Josefsen MH, Krause M, Hoorfar J (2006). Evaluation of probe chemistries and

platforms to improve the detection limit of real-time PCR. Journal of Microbiological

Methods 66(2): 206-216

Wacker MJ, Godard MP (2005). Analysis of One-Step and Two-Step Real-Time RT-PCR Using

SuperScript III. Journal of Biomolecular Techniques, 16:266-271

Page 20 of 23

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Appendix 1:

Results Questionnaire sent to each laboratory

Institution:

Operator:

Instrument used:

Results Questionnaire

RNA Extraction:

Method used:

Kit manufacturer/name of kit: Batch number: Expiry date:

Starting sample volume:

Elution volume:

Was RNA stored after extraction – how?

Comments/problems:

TaqMan RT-PCR:

Method used:

Kit manufacturer/name of kit: Batch number: Expiry date:

Reaction volume:

Cycling conditions:

Primer/probe optimisation – final reaction conditions used:

Comments/problems:

Results:

Note – not all laboratories have done all tests. Only fill in results for the tests you have done.

Threshold used:

Results AI Type A and H5 for each sample:

(H7 was done in only a few laboratories) :

Result CT value in TaqMan test: (+ or – internal control):

Was template titration was performed?

What was the limit of detection?

Comments/problems:

Please attach any additional results you may have.

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TaqMan assays Technical report

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Appendix 2:

Follow up from SCAHLS AI workshop July 2006

Recommendation for further test evaluation using AI samples from kit#2.

Update hans.heine@csiro.au 20/07/2006

Aims of Kit 2 follow up:

• Implement and evaluate use of internal controls (VIC labelled 18S rRNA control reaction)

• Determine fixed threshold value for best comparison of results between tests or

laboratories

• Determine limits of detection and set of cut-offs for reporting of TaqMan results

• Reporting of results and follow up of indeterminate TaqMan results

AI Type A TaqMan (as AAHL) [optional: AI H5 and AI H7 TaqMan]

• Order and set up 18S rRNA internal control primers and probe. Take care to adjust primers

exactly to specified concentrations. Make sure you can use VIC label on your instrument.

• Use two samples from kit No.2

(sample #7) A/shearwater/Australia/1975 H5N3 (LP) – positive control

(sample #1) A/chicken/Cambodia/1A/2004 H5N1 (HP)

• For H7 TaqMan only: (sample #2) A/emu/NSW/1997 H7N4 (HP)

• Assay conditions:

o Use primer concentration 900 nM for reverse primer

o Enzyme master mix kit (you decide and stick with it; 45 cycles).

o Applied biosystems TaqMan One-step RT-PCR master mix Part Number 4309169;

48oC/30 min; 95oC/10 min; 45x (95oC/15 sec, 60oC/60 sec), or

o Invitrogen Superscript 3 Platinum One-step Quantitative RT-PCR system

50oC/15 min; 95oC/2 min; 45x (95oC/15 sec, 60oC/30 sec) in normal mode

50oC/5 min; 95oC/2 min; 45x (95oC/3 sec, 60oC/30 sec) in fast mode on AB7500

o Internal control: plus / minus 18S rRNA

o How much RNA template used (relative to original sample)

• Evaluate the limit of detection by serial dilution of RNA from 10 -1 to 10 -7 of three different kit

No. 2 samples. Perform all tests with and without the recommended internal controls (VIC

labelled 18S rRNA control). For each of the samples perform all reactions plus and minus

18S rRNA control on the same instrument run. Perform all assays in TRIPLICATE.

• Save the results generated by the instrument computer and recalculate CT values for

different threshold levels. Calculate CT values in four different ways: Automated default

setting by instrument (please record this value for each assay). Recalculate all CT values

using three different manual threshold settings of 0.01, 0.05 and 0.1.

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• Record all TRIPLICATE CT values in table and send to AAHL (by 29 September 2006?).

Table: Record results of each test (FAM labeled) performed with (plus) and without (minus)

internal control reaction (VIC-labeled 18S rRNA) and report back to AAHL

Threshold Default value = ? Threshold = 0.01 Threshold = 0.05 Threshold = 0.1

Internal Minus Plus Minus Plus Minus Plus Minus Plus

Sample

#

control

RNA

dilution

10 -1

10 -2

10 -3

10 -4

10 -5

10 -6

10 -7

CT

triplicate

CT

triplicate

CT

triplicate

Page 23 of 23

CT

triplicate

CT

triplicate

CT

triplicate

CT

triplicate

• Analysis:

How to determine cut-off:

Dilution -1/10 Dilution -1/10 Dilution -1/10

Triplicate CT All similar pos Variation, some pos or

neg

All neg

Example only !!!! 34 38 40

Define cut-off Below 35 35 – 40 Greater 40

Report TaqMan result POSITIVE INDETERMINATE NEGATIVE

Follow up to finalise

TaqMan result

Alternative test for

indeterminates ?

None 1) Repeat test and

report result

2) Alternative sample

if practical or

necessary

(Other tests?)

None

• After comparing results from all laboratories and in consultation with AAHL each laboratory

would define their own cut-off values.

Agreement on how to report TaqMan results:

Results should be reported as POSITIVE, NEGATIVE, INDETERMINATE, or INVALID. The CT

values should NOT be included in diagnostic reports.

Suggestions and comments:

TaqMan assays Technical report

Version 300407; Checked Hans Heine

CT

triplicate


DON'T LET IT HAPPEN.

SARS, foot-&-mouth disease and avian influenza have highlighted how

vulnerable humans and animals are to disease epidemics in the global age.

Estimates of the global cost of SARS range from $10 – 100 billion. An

outbreak of foot-&-mouth in Australia, similar to the one that occurred in the

UK in 2001, could cost up to $5.8 billion in reduced livestock production

earnings alone. Avian influenza can kill up to 100% of chickens on some

farms, and some strains of the virus have infected and killed humans.

The Australian Biosecurity CRC for Emerging Infectious Disease is committed

to protecting Australia’s public health, livestock, wildlife and economic

resources by developing new capabilities to monitor, assess, predict and

respond to emerging infectious disease threats.

For more information about the Australian Biosecurity CRC, our research projects

and education and training opportunities contact:

The Communications Officer

Australian Biosecurity CRC

Building 76 Molecular Biosciences

The University of Queensland

St Lucia QLD 4072

Brisbane, AUSTRALIA

Phone +61 (0)7 3346 8864

Fax +61 (0)7 3346 8862

Email info@abcrc.org.au

Or visit our website

www.abcrc.org.au

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