Download Best Practices for Molecular Techniques in Seed Health Tests

Best Practices for Molecular Techniques in Seed Health Tests
(December 2014)
This document provides best practices on the use of molecular techniques in seed health testing
by describing recommended controls and critical points.
Given that molecular techniques are under constant development and improvement, this document
will be updated as needed to reflect the appropriate controls needed to achieve reliable results.
Introduction controls and critical points
The number and type of controls necessary for a robust seed health test using molecular
techniques depends on the application. The current use of molecular techniques in seed health
testing can be grouped into identification and detection methods. These best practices have
been established for use in conjunction with routine seed health tests. There are some
recommendations that may be essential during molecular assay development and validation, but
these have not been defined in this document.
The use of molecular techniques for identification is usually applied to a sample with a known
and/or controllable concentration of the target nucleic acid. For example a real-time PCR reaction
may be done with a cell suspension of a bacterial isolate from a dilution-plating test. The density
of the cell suspension, and therefore the concentration of the nucleic acid target can be
controlled and defined.
In the case of detection, the quantity of target in the sample is unknown. For example a real-time
PCR reaction may be done with a DNA extraction from a bio-PCR test or an RNA extraction from
seed. In the case of bio-PCR the quantity of target DNA in the sample will vary according to the
number of bacteria extracted from the seed sample and the bacterial growth during biological
amplification.
For reference, information on internal amplification controls is contained in Annex 1. Controls are
defined and their purpose described in Annex 2.
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Table 1 - Controls to include in protocols for PCR on single isolate microbial cultures for
identification
Description
Essential
Positive Process Control(s): One (1) reference isolate for
each primer target that is prepared in the same way as
the samples
x
Negative Process Control (reagent): Liquid used for
preparing microbial DNA or RNA extractions or cell
suspensions
x
Recommended
Optional
Process Controls
Negative Process Control (non-target): Non-target isolate
prepared in the same way as the positive control
x
Amplification Controls
Non Template Control: Nucleic acid-free water
x
Positive Amplification Control(s): DNA or RNA from 1 or
more reference isolate for each primer target in the case
of multiplex PCR
x
Negative Amplification Control: DNA or RNA from one
non-target isolate
x
Internal Amplification Control 1
Internal Amplification Control (IAC): Non-target DNA or
RNA sequence present in the same sample reaction tube
which is co-amplified simultaneously with the target
sequence
1
x
The use of an IAC may reduce the sensitivity of the target PCR. Non-competitive primers are
preferable to competitive primers (see Annex 1). When non-competitive primers are not available,
the IAC should be run in a separate reaction tube or well.
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Table 2 - Controls to include in protocols for Bio-PCR and direct seed wash detection assays
Description
Essential
Recommended
Optional
Process Controls
Positive Process Control: Control (media plate or seed
sample) that is known to contain the target organism and is
processed in the same way and at the same time as the
samples. The spiking control may be used for this purpose
Negative Process Control: Control that contains no target
organism or spike and is processed in the same way and at
the same time as the samples (buffer only)
x
x
Spiking Controls - Either early spiking of the test sample with a non-pathogen or split-sub-sample
spiking with the target pathogen 1, 2
Positive Extraction Control: Spiking of the target pathogen
into split subsamples of homogenised seed extracts and
treated in parallel to the test samples. The spike needs to be
detected to validate a negative sub-sample test result
OR
x
Positive Extraction Control: Early spike into the test sample
with a non-pathogen. The early spike needs to be detected in
the sample to validate a negative test result
Amplification Controls
Non-Template Control: nucleic acid-free water
x
Positive Amplification Control: DNA or RNA from 1 reference
isolate for each primer target, including non-pathogen spike
organism
x
Positive Amplification Control (nucleic acid extraction): A
spike of target DNA or RNA into a replicate PCR well for each
nucleic acid extract
x
Internal Amplification Controls 3
Internal Amplification Control (IAC): The early spike is the
source of non-target nucleic acid which is co-amplified
simultaneously with the target sequence in the same sample
reaction tube
OR
x
Internal Amplification Control: Non-target DNA or RNA is
added after nucleic acid extraction which is co-amplified
simultaneously with the target sequence in the same sample
reaction tube
1
In the case of seed wash detection tests it is preferable to use an early spike before seed extraction
to validate the complete test process.
2
In the case of bio-PCR detection tests it is preferable to use a spike in homogenised split subsamples after seed extraction to avoid problems with competition during the biological amplification
phase of the test which could occur in the case of an early spike with another organism. However,
this approach is only valid if the split subsamples are representative of the original sub-sample.
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The use of an IAC may reduce the sensitivity of the target PCR. Non-competitive primers are
preferable to competitive primer (see Annex 1). When non-competitive primers are not available, the
IAC should be run in a separate reaction tube or well.
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Table 3 - PCR critical points required for a valid molecular assay result
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Positive control reaction results should contain the specific PCR product without non-specific
products
2
Negative controls reaction results should not contain the specific PCR product
3
In the case of an internal amplification control, the expected product should be observed in order
to validate the test result
4
In the case of real-time PCR, the Ct values should be similar between reactions with similar
concentrations of target DNA. Significant differences indicate that the PCR was not optimal. This
may result in false positive reactions or PCR inhibition
During the design of molecular methods for ISHI protocols, certain points should be indicated as
critical in the protocol. These identified critical points cannot be modified. Other parts of molecular
protocols can be considered as guidelines and modified according to specific laboratory conditions.
Table 4 - Critical and non-critical items in established molecular assays
Detail
Status
Comments
Detection target (pathogen)
primer sequences
Non-changeable
Detection target (pathogen)
probe sequences
Non-changeable
Probe modifications
Non-changeable
Probe modifications (e.g., MGB, LNA) can
significantly change Tm values and specificity of
probes
Internal amplification
control PCR primers
Changeable
Internal control PCR primer sequences should be
proposed, but flexibility should allow laboratories to
use internally validated control primers or commercial
kits with internal control primers. Possible
interactions with the specific pathogen primers/probe
should be investigated by the laboratory
PCR conditions
(temperature; time)
Changeable
Reaction mix (Taq, buffer,
MgCl2, dNTPs) supplier and
concentrations
Changeable
Probe dyes (fluorophore,
quencher)
Changeable
PCR product identification
(gel-electrophoresis or
SYBR green)
Changeable
PCR machine
Changeable
Probe cannot be added without additional validation
The impact on the sensitivity of the detection method
due to protocol changes needs to be verified
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Annex 1: Internal Amplification Control (IAC)
Definition: An IAC is a non-target nucleic acid sequence present in the same sample reaction tube
which is co-amplified simultaneously with the target sequence.
Multiplexing PCR reactions of IAC and target may lead to a decrease of sensitivity of the target
PCR. In that case, it is preferred to use an IAC in a simplex PCR reaction. Amplification of the IAC
indicates that the PCR matrix was suitable for PCR to occur. In a PCR without IAC, a negative
response can mean that no target sequence was present in the reaction or that the reaction was
inhibited due to a malfunction of the thermal cycler, incorrect PCR mixture, poor polymerase
activity, presence of inhibitors in the sample matrix. There are two types of IAC: competitive and
non-competitive.
Competitive IAC
Non-competitive IAC
Primers
same primers for target and IAC
different primers for IAC and target
product length or sequence
IAC identification
product length or sequence
use of different dye-labeled probes
in real time PCR
difference in melting peaks
Critical factors
concentration IAC
concentration IAC
nucleotide composition IAC
size of the IAC
Competition between
IAC & target
yes, for primers, oligonucleotides
and DNA polymerase
yes, for oligonucleotides and DNA
polymerase
Drawbacks
lower detection limit due to the
competition by IAC
for each target optimization and
production of IAC is needed
production of IAC is time
consuming, labor intensive (cloning
step required for IAC production)
amplification of noncompetitive
sequences may not accurately reflect
amplification of the primary target due to
the differences in the primer sequences
risk of undesired interactions of multiple
primers
can be used for many different assays
in the same laboratory (universally)
Advantages
Specification IAC
Example IAC
Commercial IAC kits
size IAC > target
Modified target IAC (mimic) by
deleting, inserting or modifying
sequences between the recognition
primer sites. A hybridization probe
can be designed which detects IAC
specifically.
insert the (modified) target
sequence in a plasmid
construction of IAC with completely
designed nucleotide sequence
Synthetic DNA (IAC plasmid DNA) or
another gene (e.g. encoding rRNA)
which is present in any microorganism
and in higher copy number than the
principal target gene.
primers specific to conserved
sequences of 16S and 23S ribosomal
DNA
add a non relevant microorganism,
which is usually not found in the sample
type to be tested
exogenous source of DNA template
PrimerDesign Ltd
www.primerdesign.co.uk
1. J. Hoorfar, N. Cook, B. Malorny, M. Wagner, D. De Medici, A. Abdulmawjood and P. Fach. 2003.
Making Internal Amplification Control Mandatory for Diagnostic PCR. Journal of Clinical
Microbiology, 41:12.
2. J. Hoorfar, B. Malorny, A. Abdulmawjood, N. Cook, M. Wagner, P. Fach. 2004. Practical
Considerations in Design of Internal Amplification Controls for Diagnostic PCR Assays. Journal of
Clinical Microbiology, 42:5.
3. Anonymous. 2002. Microbiology of food and animal feeding stuffs. Polymerase chain reaction
(PCR) for the detection of food-borne pathogens. General method specific requirements (EN ISO
22174). International Organization for Standardization, AFNOR, Paris, France.
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4. QCMD (Quality Control for Molecular Diagnostics) http://www.qcmd.org/
QCMD is an independent International External Quality Assessment (EQA) / Proficiency Testing (PT)
organisation. It provides a wide-ranging quality assessment service primarily focused on molecular
infectious diseases to over 2000 participants in over 100 countries. QCMD is dedicated to advancing
the quality of molecular diagnostics through External Quality Assessment (EQA), Proficiency Testing
(PT) and other supporting quality initiatives
Annex 2: Definition of Controls used in PCR
Positive Process Control: These are additional test samples that are known to contain the target
organism and are processed in the same way and at the same time as the suspect samples. For
microbial isolate identification (Table 1), this can be a freshly prepared suspension of reference
isolate. For microbial detection assays, this can be a known infected sample, spiked seed
sample, or spiked buffer without seeds. The purpose of this control is to verify the quality of the
materials and methods used in the pathogen extraction as well as nucleic acid (DNA or RNA)
extraction process for all samples run simultaneously by demonstrating detection of known target
in the same process cycle within defined process parameters.
Negative Process Control (non-target): Freshly prepared suspension of non-target organism
used for spiking in a detection procedure in which an early spike with a non-pathogen is applied.
The purpose of this control is to verify the quality of the materials and methods used in the
pathogen extraction as well as nucleic acid extraction process for all samples run simultaneously
by demonstrating detection of an inoculated non-pathogen in the same process cycle within
defined process parameters.
Negative Process Control (reagent): Control that contains no target organism or spike and is
processed in the same way and at the same time as the samples (buffer only). The purpose of
this control is to verify the quality of the materials and methods used in the pathogen extraction
as well as nucleic acid extraction process by demonstrating no detection (and/or below a
defined threshold) of target or spike DNA or RNA.
Spiking Control (created by spiking samples with a known organism): This can be performed by
either a) an “early spike” of the test samples with a non-pathogen that resembles the target
pathogen but is distinguishable or b) spiking of the target pathogen into one half of a split
subsample of homogenized seed extract. The purpose of this control is to verify the quality of the
materials and methods used in the pathogen extraction as well as DNA or RNA extraction
process for the specific sample by demonstrating detection of an inoculated organism within
defined process parameters. The spike needs to be detected to validate a negative sample
process result.
Non-Template Control (NTC): Containing all PCR reagents but no target DNA, RNA or IAC
nucleic acids. This is also called a blank. Water or buffer controls can serve as the NTC. The
purpose of this control is to verify the quality (i.e., nucleic acid-free) of the materials used in the
PCR process by demonstrating no detection of target DNA or RNA when it has not been added.
Positive Amplification Control: DNA or RNA of one or more reference isolate for each primer
target. The purpose of this control is to verify the quality of the materials and methods used in the
PCR process by demonstrating detection of a standardized nucleic acid target within defined
process parameters.
Negative Amplification Control: DNA or RNA of one or more non-target reference isolate. The
purpose of this control is to verify the quality (specificity) of the materials and methods used in
the PCR process by demonstrating non-detection of standardized non-target DNA or RNA.
Internal Amplification Control (IAC): Non-target DNA or RNA sequence present in the same
sample reaction tube which is amplified simultaneously with the target sequence. Note: the use
of an IAC may reduce the sensitivity of the target PCR. There are several options possible: a)
IAC (DNA or RNA) is added after nucleic acid extraction, b) IAC (cells, particles) is added before
nucleic acid extraction (“late spike”) or c) “early spike” (see: “spiking control” above). The
purpose of this control is to demonstrate the quality of the materials used in PCR to indicate
amplification conditions were met. This control validates a negative sample PCR result.
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