aavv-1 screening real time pcr · qiagen quantifast rt mix 0.25 l 4.3.3 add reagents as above. make...

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Avian Influenza Community Reference Laboratory

AAvV-1 screening real time PCR

This protocol is a copy of the standard operating procedure

used by the avian influenza CRL at the Animal and Plant Health

Agency. If you have any technical queries

please contact [email protected]


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Contents

1. INTRODUCTION ................................................................................................ 3

1.1 PURPOSE/SCOPE OF THIS SOP .............................................................................................. 3 1.2 BACKGROUND INFORMATION ................................................................................................... 3

2. SAFETY ........................................................................................................................................ 4

3. MATERIALS ................................................................................................................................. 4

3.1 DOCUMENTATION AND SOFTWARE ........................................................................................... 4 3.2 CHEMICALS AND REAGENTS .................................................................................................... 4 3.3 EQUIPMENT ............................................................................................................................ 5

4. PROCEDURE/METHOD .............................................................................................................. 6

4.1 TEST RELIABILITY ................................................................................................................... 6 4.2 METHOD - PREPARATION OF STANDARDS ................................................................................. 6

4.3 .. PREPARATION OF PCR MASTERMIX 6 4.4 ADDITION OF SAMPLES AND CONTROLS 7 4.5 RUNNING THE REACTIONS 8

5. RESULTS ..................................................................................................................................... 9

5.1 ANALYSIS AND DISPLAY OF RESULTS 10

5.2 INTERPRETATION OF RESULTS 11

6. CONTINGENCIES ................................................................................................................ 12

7. REFERENCES ........................................................................................................................... 12


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1. INTRODUCTION

1.1 Purpose/Scope of this SOP

1.1.1 To rapidly detect avian avulavirus type-1 (AAvV-1), formerly avian

paramyxovirus type 1 (APMV-1), from RNA extracted from chick embryo

amplified samples and clinical material including avian tissues, swabs

and fluids.

1.2 Background information

1.2.1 Real-time polymerase chain reaction (PCR) is a technology that

combines DNA amplification with the fluorescent detection of the

products in a single tube. In the case of RNA viruses an extra reverse

transcriptase step is required to convert the RNA into cDNA. This test

combines the reverse transcription, PCR and detection in a single tube.

This format is highly beneficial as it removes the significant contamination

risk caused by opening tubes for post-PCR manipulation. It is also less

time consuming than gel based analysis and can supply a quantitative

result.

1.2.2 The current detection method is based upon fluorescent probe

technology in which fluorescence increases proportional to an increase in

product. Fluorescence is monitored during each PCR cycle to provide an

amplification plot that can be analysed to confirm the presence or

absence of AAvV-1 RNA. Real-time PCR machines analyse raw

fluorescence data and produce a cycle threshold (Ct) value for each

sample. The cycle at which the fluorescence increases above the critical

threshold is reported as the Ct value. The critical threshold is an arbitrary

fluorescence value that can be manipulated by the operator but must be

set in the exponential phase of the reaction. The accuracy of the ct value

is dependent upon the fluorescent reading of the raw data and the

fluorescent value for the critical threshold.

1.2.3 The PCR is based upon a 160bp, conserved region of the AAvV-1 L

polymerase gene, in which two probes are included, to ensure that all six

lineages of AAvV-1 can be detected.


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2. SAFETY

2.1 It is your laboratory’s responsibility to ensure all work described in this

protocol is conducted to a high safety standard. This includes an

awareness of risks relating to e.g. dangerous or toxic chemicals,

potentially hazardous procedures etc. Local safety rules in your

laboratory should be understood by all relevant members of staff.

3. MATERIALS

3.1 Documentation and software

3.1.1 All details of AAvV-1 rRT-PCR test procedures carried out should be

recorded.

3.1.2 All scientists who are trained to perform this AAvV-1 rRT PCR on a real-

time PCR platform must be sufficiently familiar with the accompanying

relevant software in order to run an experiment.

3.2 Chemicals and reagents

3.2.1 Primers and probes – Invitrogen for MGB probes

NDF GAG CTA ATG AAC ATT CTT TC

NDR AAT AGG CGG ACC ACA TC TG

LproMGB 6FAM] CCA ATC AAC TTC CC [MGBNFQ] (e.g Life

Technologies cat No 4316034

LproMGB2 [VIC] AAT AGT GTA TGA CAA CAC [MGBNFQ]

3.2.2 Reagents:

Molecular grade water – any supplier

Qiagen Quantifast Probe RT-PCR+ROX VialKit (400) Cat No 204554

3.2.3 AAvV-1 rRT-PCR Controls No template control

Include at least one no template control. Label this as “NTC” in the

instrument software. This is to control for contamination of Mastermix.


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3.2.4 Negative extraction control

Include at least one negative extraction control (autoclaved deionised

water) per run. This should be labelled as FAM Negative extraction

control in the instrument software. This controls for cross contamination

during handling of the samples

3.2.5 Positive extraction control

Include in each run a control from, for example, Ulster (avirulent) AAvV-1,

diluted to a pre-determined Ct value (between 28-32) upon extraction.

This should be labelled as “FAM positive extraction control” in the

instrument software. This controls for the efficiency of the RNA extraction.

Refer to Appendix 1 for preparation of new batches of positive controls.

3.2.6 Standard RNA controls

Viral RNA is extracted from egg allantoic fluid, where an appropriate

AAvV-1 isolate has been propagated and grown in embryonated fowls’

eggs (EFEs).

3.3 Equipment

3.3.1 Microcentrofuge tubes (1.5ml and

0.5ml)

Realtime PCR instrument (eg.

MX3000P)

-70C or lower freezer -18C or lower freezer

Sterile RNAse free tips with

aerosol barrier

Microcentrifuge

Pipettes Vortex mixer

Polystyrene bijou or Universal Ultracentrifuge

Real-time instrument plastic ware Plate sealers

Qiagen Biorobot Universal Foil


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4. PROCEDURE/METHOD

4.1 Test Reliability

4.1.1 A positive control is included in each extraction and PCR test. The Ct

values obtained from the extraction control and standards should fall

within the range stated in the results section below. If not, the reason

behind an out of range result being obtained should be investigated.

The Ct values obtained from the positive extraction control and standards

% efficiency values are compared over time to identify trends that may

indicate poor performance of any component of the test. Once a trend is

identified, its cause should be investigated.

4.2 Method - Preparation of standards- see Appendix 1

4.2.1 Stocks of Ulster [ ] are grown according to internal methods.

4.2.2 RNA is prepared in batches with a designated arbitrary value of 107

and stored at -70C. Take out an aliquot and thaw. Carefully prepare

ten-fold dilutions of RNA in DEPC water as follows:-

Ten-fold dilution Designated value

(label tubes with these values)

10-1 106

10-2 105

10-3 104

10-4 103

10-5 102

10-6 101

Mix each dilution by agitation / flicking and briefly centrifuge. Make

sufficient of each dilution so that enough is made to use for all the AAvV-

1 screening rRT-PCR runs for that day. Store the aliquots on ice.

Discard at the end of the working day.

4.3 Preparation of PCR mastermix

4.3.1 Preparation of PCR master mix and loading of real-time plate/strip(s) to

be carried out in a PCR clean room.


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4.3.2 Reagent Volume x1

Molecular grade water (i.e. RNAse-free) 2.75l

(x2) Qiagen QuantiFast Probe RT PCR mastermix (w/o ROX) 12.5l

ROX dye solution 0.5l

NDF (12.5M in molecular grade water) 1l

NDR (12.5M in molecular grade water) 1l

LproMGB (5M in TE buffer) 1l

LproMGB 2 (5M in TE buffer) 1l

Qiagen QuantiFast RT mix 0.25l

4.3.3 Add reagents as above. Make up master mix sufficient for the number of

samples to be tested. Smaller volumes can be made in a 1.5ml micro-

centrifuge tube, for larger volumes use either a bijou or a universal.

4.3.4 Thoroughly mix the master mix. For micro-centrifuge tubes pulse spin the

tube to remove any bubbles. For bijoux/universals no spin is needed as

they are nonstick.

4.3.5 Aliquot 20l of master mix per well of the real-time plate/strip(s). Be sure

to use an appropriate plastic ware for the real-time machine you will be

using.

4.3.6 Loosely place the plate caps on the plate or cover with plate sealer or foil.

4.3.7 Bring the plate/strip(s) out of the clean room for the addition of sample

RNA and controls.

4.4 Addition of samples and controls

4.4.1 RNA is extracted from the samples according to commercial kit

instructions

4.4.2 In order to minimise the risk of contamination, the different types of

controls and test specimens should be added in the following

chronological order. It is important to keep wells covered after addition of

control / sample, and also to add in such a manner to minimise any carry-

over contamination risk into open wells to which control / sample has not


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yet been added. Change gloves frequently and do not hold tips

containing RNA above the incorrect wells.

Referring to plate layout, add 5l DEPC water as a no template control

(NTC), Add 5l of the negative extraction control, Replace caps on tubes.

Then add the sample RNA to the master mix. Replace caps on tubes.

4.4.3 Add the real time PCR RNA standards (from weakest to strongest) and

positive RNA extraction control to appropriate wells based on your

worksheet layout.

Once RNA is added, fit caps to all wells.

It is important that the caps are fitted firmly and correctly onto the wells

before the plate is inserted into the real time machine.

4.4.4 Where necessary, briefly spin down plate/strip(s) contents in a centrifuge.

4.4.5 Real-time plate/strip(s) should be kept at on ice or at +4C until ready to

test.

4.5 Running the reactions

4.5.1 The instructions below are for the Agilent MX3000 PCR machine and

indicate the settings that should be used.

Open a new file on the machine and use the real time quantitative PCR

(multiple standards) option for the experiment type.

N.B: ’Filter Gain Setting’ - After servicing or repair this setting is often

changed back to the factory default of FAM x8. On return to APHA the

MRM team routinely correct the filters for our use Cy5 ROX and HEX

should all be set to x1, FAM should be set to x2. Please see example

below. If prompted or you wish to check the filter settings, it is possible

in the MXpro software to change the ‘Filter Set Gain Settings’ – click

Instrument, then filter Set Gain settings.


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Following the plate layout, assign well type to each well. Unknown

samples are designated as such. AIV RNA standards (10-fold dilution

series) are designated as standards and the value entered corresponds

to its designated value (see para 4.2.3).

4.5.2 Select the correct filters – ROX, FAM and HEX.

Set ROX as the reference dye

..

4.5.3 Label the controls as follows; Mastermix/H2O only=NTC, negative

extraction= FAM negative control, positive extraction controls=FAM

positive control, positive RNA control=Standard

4.5.4 The cycling conditions for this test are

50C for 10 minutes

95C for 5min

followed by 40 cycles of

95C for 10 seconds

50C for 30 seconds

72C for 30 seconds

Fluorescence is read at the end of the 50C for 30 seconds annealing

step.

4.5.5 Select the start run after 20 minutes lamp warm up option when

initiating the PCR run. It is recommended that the lamp is warmed up

before preparing the Mastermix.

5. RESULTS

The analysis procedure described below is for the MX-Pro software and

should be adapted for the software being used.


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5.1 Analysis and display of results

For each reporter dye in every well the raw fluorescence data will be

plotted by the software. The raw data will be automatically analysed by

the accompanying relevant software to create a critical threshold and use

this to report any Ct values. Users must be aware of how to access raw

data from appropriate real-time PCR software. The steps to interpreting

results on MXPro software are:

If the raw fluorescence is above 35000 the accuracy of the readings is

reduced. In this case check the filter gain settings to ensure they are set

to the correct values as defined above. The baseline signal for all the

amplification plots must also be above 3000 RFU.

5.1.1 To analyse the data, select the ‘Analysis’ section button and select the

wells to be examined in the Analysis/Setup window.

5.1.2 To view the results, click on the ‘Results’ tab in the ‘Analysis’

section and view the amplification plots.

5.1.3 Examine the critical threshold using either dR (baseline-corrected raw

fluorescence) when no background reference dye (e.g. ROX) has been

used and dRn (baseline-corrected normalized fluorescence) when a

background reference dye has been used. The critical threshold will be

set automatically by the software but you must check that the software

algorithm has not set the bar too low or too high. The critical threshold

must be set in the exponential phase of the reaction above the

background fluorescence at a point where the amplification of the

controls is vertical on a logarithmic scale. Finally check the reported CT’s

for the controls as below.


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5.1.4 Analyse the data by first checking the fluorescence threshold cut-off and

comparing the results obtained for the negative ‘No template control’ and

positive extraction controls.

5.2 Interpretation of results

5.2.1 For a valid test the AAvV-1 rRT-PCR controls must meet the following

conditions:

1. No template control – “No Ct” or ct must be 37.

2. Negative extraction control – “No Ct” or ct must be 37.

3. [ ] Positive extraction controls - Ct value of the extraction control must be

within two Ct of the predetermined batch Ct value. For example if the

predetermined Ct value of a batch of Ulster was 30 [ ] then on a valid test the

Ulster Ct value would be between 28-32 [ ].

5.2.2 A dilution series of positive RNA controls as standards: Construct a

standard curve with the positive standards Ct values. The curve should

comply with the following criteria:

Efficiency: >80% and <120%

Slope between –3.1 and –3.8


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R2 value: >0.980

These tolerances are acceptable for obtaining a qualitative rRT-PCR

result in the context of testing submission cases, outbreak specimens

and screening work, and do not require accurate quantification of the

neat AAvV-1 RNA used the construct the dilution series.

5.2.3 If the test fails the above parameters it must be repeated. Test failures

may arise from a number of factors including suboptimal extraction of the

positive control (and therefore test) specimens, the positive extraction

control may have degraded if stored incorrectly e.g. 4oC, degradation of

probes through repeat freeze-thawing, pipetting errors when preparing

the RNA standard dilution series etc.

5.2.4 A positive sample produces a Ct value of 37 with either of the probe

dyes used (HEX or FAM) with the lowest Ct value being reported.

Negative results produce “No Ct”. A Ct value of 37 is an inconclusive

result and requires repeat testing for confirmation. A ‘late’ Ct value of 37

on repeat testing is reported as negative.

5.2.5 If the ‘late’ Ct value (37) has a logarithmic/sigmoidal character where

clear final fluorescence values are observed, then contamination of the

no template control wells with AAvV-1 RNA may be considered. It is also

possible that such very late Ct signals may occasionally occur spuriously.

Repeat the AAvV-1 rRT-PCR assay.

6. CONTINGENCIES

6.1 Any queries regarding the test performance, troubleshooting and/or

discussion of positive or negative result can be referred to the test and

disease consultants.

7. REFERENCES

Chad M. Fuller, Lina Brodd, Richard M. Irvine, Dennis J. Alexander and

Elizabeth W. Aldous. 2010. Development of an L gene real-time Reverse-

Transcription PCR assay for the detection of Avian Paramyxovirus type 1

RNA in clinical samples. Archives of Virology (2010) 155:817-823 DOI:

10.1007/s00705-010-0632-1


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8. Appendix 1

Preparation of TaqMan®real-time RT- PCR positive extraction controls and RNA

standards

These can be prepared from either live or inactivated egg-grown antigen and can be

supplied either as an aliquot of freeze-dried material or an aliquot from a wet ‘antigen

stock.

NB - For live antigen follow safety procedures in place for the virus type being

produced. Work at the appropriate containment level for the virus being used – refer

to the virus categorisation risk document

To prepare positive extraction controls

1. If the antigen is supplied freeze-dried, reconstitute the vial with 1ml molecular grade

water. Ensure the solution is mixed thoroughly by gentle vortexing.

2. Prepare a 10-fold dilution series of the antigen in PBS e.g. from 10-1 - 10-6 dilutions

inclusive.

3. Freeze 200 µl aliquots of each dilution at -70 ºC.

4. Thaw-out an aliquot of each dilution and extract RNA from each aliquot either

manually or on the Bio-robot according to procedure for RNA extraction from

biological samples or Extraction of nucleic acids from swabs using the Qiagen

BioRobot Universal.

5. Test the RNA from each member of the dilution series by the appropriate real-time

PCR assay. Choose the optimal 10-fold dilution from the resulting Ct values. The

acceptable Ct value range is documented in the relevant SOP for the real-time PCR

assay used.

6. Prepare the chosen dilution of the antigen in bulk using PBS, assign a batch

number, dispense into 200µl aliquots, label and freeze at -70 ºC.

7. Remove an aliquot from the freezer, extract the RNA and test by the appropriate

real-time RT-PCR to confirm the Ct values from the dilution fall within the required

range.

To prepare RNA PCR standards

1. If the antigen is supplied freeze–dried, reconstitute the vial with 1ml molecular

grade water. Ensure the solution is mixed thoroughly by gentle vortexing.

2. Manually extract the RNA from the whole reconstituted antigen from 1ml of wet

stock according to the procedure for RNA extraction from biological samples.


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3. Pool together the RNA extractions.

4. Freeze a 5µl aliquot at-70 ºC

5. Thaw this aliquot and prepare a 10-fold dilution series in molecular grade water

from as described above from 10-1 - 10-7

6. Test the dilution series by the appropriate real-time RT-PCR. Test the current ‘in-

use’ RNA standard batch in parallel on the same PCR test run.

7. Directly compare the results of the two batches. The Ct values and test efficiencies

should be similar i.e. Ct values +/- 2.0 per corresponding dilution. If there is an

apparent significant difference, adjust the dilution of the new batch of RNA (eg.

make a 1/10 dilution) and repeat stages 3-7 above.

8. Once the required dilution is achieved, prepare 5µl aliquots, assign a batch number,

label the tubes and store at -70 ºC.

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