Assessment of the laboratories performing molecular biology testing

17-19 September 2019

Ministry of Economy and Infrastructure

Support to the Quality Infrastructure
Framework within a DCFTA Context in the
Republic of Moldova
EuropeAid/138295/DH/SER/MD
Objectives of the course
Molecular approaches in the diagnosis of human diseases
Detection of pathogens
Detection of genetic conditions
Analysis of microbiota
Qualitative and quantitative tests
Validation vs verification
Uncertainty
Real Time PCR 
 Real Time PCR 

A real-time polymerase chain reaction (real-time PCR),

also known as quantitative polymerase chain reaction
(qPCR), is a laboratory technique of molecular biology
based on the polymerase chain reaction (PCR). It
monitors the amplification of a targeted DNA molecule
during the PCR (i.e., in real time), not at its end, as in
conventional PCR.
 Real Time PCR 

Real-time PCR can be used quantitatively (quantitative

real-time PCR) and semi-quantitatively (i.e.,
above/below a certain amount of DNA molecules)
(semi-quantitative real-time PCR).
 Real Time PCR 

Real-time PCR is carried out in a thermal cycler with the

capacity to illuminate each sample with a beam of light
of at least one specified wavelength and detect the
fluorescence emitted by the excited fluorophore. The
thermal cycler is also able to rapidly heat and chill
samples, thereby taking advantage of the
physicochemical properties of the nucleic acids and
DNA polymerase.
 Real Time PCR 

The PCR process generally consists of a series of

temperature changes that are repeated 25 – 50 times.
These cycles normally consist of three stages: the first,
at around 95 °C, allows the separation of the nucleic
acid's double chain; the second, at a temperature of
around 50-60 °C, allows the binding of the primers with
the DNA template; the third, at between 68 - 72 °C,
facilitates the polymerization carried out by the DNA
polymerase. 
 Real Time PCR 

Real-time PCR technique can be classified by the

chemistry used to detect the PCR product, specific or
non-specific fluorochromes:

 Non-specific detection: Real-time PCR with double-

stranded DNA-binding dyes as reporters

 Specific detection: fluorescent reporter probe

method
 Real Time PCR 
Non-specific detection: Real-time PCR with
double-stranded DNA-binding dyes as
reporters
A DNA-binding dye binds to all
double-stranded (ds) DNA in PCR,
causing fluorescence of the dye. An
increase in DNA product during PCR
therefore leads to an increase in
fluorescence intensity measured at
each cycle.
Real Time PCR 
TAQMAN Probe
 Real Time PCR 

Non-specific detection: Real-time PCR with double-

stranded DNA-binding dyes as reporters

In real-time PCR with dsDNA dyes the reaction is

prepared as usual, with the addition of fluorescent
dsDNA dye. Then the reaction is run in a real-time PCR
instrument, and after each cycle, the intensity of
fluorescence is measured with a detector; the dye only
fluoresces when bound to the dsDNA (i.e., the PCR
product).
 Real Time PCR 

Non-specific detection: Real-time PCR with double-

stranded DNA-binding dyes as reporters

This method has the advantage of only needing a pair

of primers to carry out the amplification, which keeps
costs down; multiple target sequences can be
monitored in a tube by using different types of dyes.
Aspecific
product
 Real Time PCR 

Specific detection: fluorescent reporter probe method

Fluorescent reporter probes detect only the DNA

containing the sequence complementary to the probe;
therefore, use of the reporter probe significantly
increases specificity, and enables performing the
technique even in the presence of other dsDNA.
 Real Time PCR 

Specific detection: fluorescent reporter probe method

Using different-coloured labels, fluorescent probes can

be used in multiplex assays for monitoring several
target sequences in the same tube. The specificity of
fluorescent reporter probes also prevents interference
of measurements caused by primer dimers, which are
undesirable potential by-products in PCR.
 Real Time PCR 
Specific detection: fluorescent reporter probe method

1. The PCR is prepared as usual and the reporter probe

is added.
2. As the reaction commences, during the annealing
stage of the PCR both probe and primers anneal to the
DNA target.
5’ REPORTER (R): High Energy Fluorochrome
3’ QUENCHER (Q): Low Energy Fluorochrome

Q switch off R!!

 Real Time PCR 

Specific detection: fluorescent reporter probe method

3. Polymerisation of a new DNA strand is initiated from

the primers, and once the polymerase reaches the
probe, its 5'-3'-exonuclease degrades the probe,
physically separating the fluorescent reporter from the
quencher, resulting in an increase in fluorescence.
High specificity
 Real Time PCR 

Specific detection: fluorescent reporter probe method

4. Fluorescence is detected and measured in a real-time

PCR machine, and its geometric increase corresponding
to exponential increase of the product is used to
determine the quantification cycle (Cq) in each
reaction.
 Pros and Cons
SYBR GREEN TAQMAN

PROS: PROS:
Simple method Simple method
Flexible Highly specific
Not expensive Allow calculation of Delta Ct
Multiplexing possible
CONS:
Aspecific: the fluorochrome binds to all CONS:
double helix, including primers dimers Expensive
Requires time-consuming validation Not flexible
processes
Requires a standard curve and melting
analysis
Real Time PCR 
Real Time PCR 
Real Time PCR 
 METHOD VALIDATION 
Definitions: 

5.5.1.3 Validation of examination procedures

The laboratory shall validate examination procedures derived from the
following sources:
a) non-standard methods;
b) laboratory designed or developed methods;
c) standard methods used outside their intended scope;
d) validated methods subsequently modified.
[UNI CEI EN 15189:2012]
 METHOD VALIDATION 
Definitions: 

5.5.1.2 Verification of examination procedures

Validated examination procedures used without modification shall be subject to
independent verification by the laboratory before being introduced into routine
use. .
The laboratory shall obtain information from the manufacturer/method
developer for confirming the performance characteristics of the procedure.
The independent verification by the laboratory shall confirm, through obtaining
objective evidence (in the form of performance characteristics) that the
performance claims for the examination procedure have been met.
[UNI CEI EN 15189:2012]
 METHOD VALIDATION 
Definitions: 

5.5.1.2 Verification of examination procedures

The performance claims for the examination procedure confirmed during the
verification process shall be those relevant to the intended use of the
examination results.
The laboratory shall document the procedure used for the verification and
record the results obtained. Staff with the appropriate authority shall review the
verification results and record the review.
[UNI CEI EN 15189:2012]
 IN BRIEF 

Validation
• Lab-developed methods
• Non-standard methods
• Standard methods outside their intended scope
• Modifications of standard methods

Verification
• Standard methods
• Methods validated by recognized organizations
Method features and their meaning

Sensitivity and Specificity

Limit of detection (LOD)
Limit of quantification (LOQ)
Linearity and measuring range
Precision
Accuracy
Robustness
Unicertainty
 Validation parameters
Qualitative methods
Sensitivity and Specificity
Limit of detection (LOD)
Accuracy (Accordance)
Robustness
Uncertainty
Quantitative methods
Sensitivity and Specificity
Limit of detection (LOD)
Limit of quantification (LOQ)
Linearity and measuring range
Precision
Accuracy
Robustness
Uncertainty
Traditional probabilistic measures of sensitivity and
specificity
a) FALSE NEGATIVE (TYPE I ERROR) RATE: Percent rejection of true
condition.
FNR = [NFN /(NTP +NFN)]
b) FALSE POSITIVE (TYPE II ERROR) RATE: Percent failure to reject false
condition.
FPR = [NFP /(NFP +NTN)]
c) SENSITIVITY: Percent confirming a true condition.
Se = [NTP /(NTP +NFN)]
d) SPECIFICITY: Percent rejecting a false condition.
Sp = [NTN /(NFP +NTN)]
e) POSITIVE PREDICTIVE VALUE: Percent indicating condition true that are
correct.
Ppv = [NTP /(NFP +NTP)]
f) NEGATIVE PREDICTIVE VALUE: Percent indicating condition false that are
correct.
Npv = [NTN /(NFN +NTN)]
When engaging in qualitative analysis,
“the paradigm of yes/no conclusions is
useful for describing and quantifying the
accuracy with which molecular biology
disciplines can provide answers. In such
situations, results from analyses for which
the truth is known can be classified in a
two-way table.
   True situation  
above (or below the total sum
equal to) limit (a + b) and
the limit (c + d)
Test result positive 125 20 145
negative 25 130 155
  total 150 150 300
   True situation  
above (or below the total sum
equal to) limit (a + b) and
the limit (c + d)
Test result positive (a) (b) a+b
negative (c) (d) c+d
  total (a + c) (b + d) a+b+c+
d
Sensitivity = a/a+c Specificity = d/b+d
False negatives = c/a+c False positives = b/b+d
Positive predicted values Positive predicted values =
= a/a+b d/c+d

Sensitivity = 0.83 Specificity = 0.87

False negatives = 0.17 False positives = 0.13
Positive predicted values = Positive predicted values =
0.86 0.84
Bayes' theorem
In probability theory and statistics, Bayes' theorem describes the
probability of an event, based on conditions that might be related
to the event.

where:
P(A /e) is the probability of the occurrence of A given the event e
P(A) is the probability “a priori” of the occurrence of A
P(¬A) is the probability “a priori” of the absence of A
P(e / A) is the probability of the event e given the occurrence of A
P(e /¬A) is the probability of the event e given the absence of A,
i.e. the probability of a false positive
False negatives

False positives
 Validation parameters
Qualitative methods
Sensitivity and Specificity
Limit of detection (LOD)
Accuracy (Accordance)
Robustness
Uncertainty
Quantitative methods
Sensitivity and Specificity
Limit of detection (LOD)
Limit of quantification (LOQ)
Linearity and measuring range
Precision
Accuracy
Robustness
Uncertainty
Sensitivity should be referred to
true positive / true negative samples

How to define a true positive?

Sensitivity should be referred to
true positive / true negative samples

The case of Trichomonas vaginalis

CPLM medium
=
Gold standard
The concept of specificity in molecular diagnostic
methods of infectious diseases is related to:

Inclusivity Exclusivity
Inclusivity: the test should be able to
detect all species/subspecies/variants
of the pathogen we want to detect
Exclusivity: the test should NOT be
able to detect any other
species/subspecies/variants
of related or unrelated pathogens
other then the target.
LOD – LIMIT OF
DETECTION
LOD – LIMIT OF
DETECTION

Target DNA
copy number
LOD – LIMIT OF
DETECTION
 Real Time PCR 

Real-time PCR can be used quantitatively (quantitative

real-time PCR) and semi-quantitatively (i.e.,
above/below a certain amount of DNA molecules)
(semi-quantitative real-time PCR).
 Cut-off 

Analytical sensibility and analytical specificity are

directly related to LOD

Diagnostic sensibility and diagnostic specificity

are related to a quantitative value known as cut-off
 Cut-off 

There are several methods to calculate the cut-off

Cut-offs can be selected as those values that define

a high DSe (e.g. inclusion of 99% of the values from
infected animals), and a high DSp (e.g. 99% of the
values from uninfected animals)
 Cut-off 

Once a cut-off value is determined, the test can

not retained as qualitative but as semi-
quantitative.

Therefore the method must undergo semi-

quantitative validation processes
 Cut-off 

When a cut-off value has been determined and it is

used to define the presence or the absence of the
DNA of a pathogen, the the test can not retained
as qualitative but as semi-quantitative.
I.E. A QUANTITATIVE TEST WHOSE RESULT IS
EXPRESSED IN A QUALITATIVE MANNER.
 REPEATABILITY 

When a cut-off value has been determined and it is

used to define the presence or the absence of the
DNA of a pathogen, the the test can not retained
as qualitative but as semi-quantitative.
THEREFORE A LIMIT OF REPEATABILITY CAN BE
EVALUATED, AND USED TO VERIFY REPEATABILITY
IN DUPLICATE ANALYSES
 REPEATABILITY 

THE LIMIT OF REPEATABILITY CAN BE EVALUATED

AS FOLLOWS:

Where t is the value of Student’s distribution

and sr is the Standard Deviation
 REPEATABILITY 

THE FOLLOWING REQUIREMENT MUST BE MET:

|X1 – X2| ≤ r

Where X1 and X2 are the two values obtained in the

duplicate analysis (i.e. the two values of Ct)
LOQ – LIMIT OF
QUANTIFICATION
LOQ – LIMIT OF
QUANTIFICATION
LOQ – LIMIT OF
QUANTIFICATION
 ABSOLUTE
QUANTIFICATION

The slope is a
measure of
the efficacy of
The correlation coefficient is a measure of the reliability of the reaction
the standard curve
ABSOLUTE
QUANTIFICATION

Efficacy
ABSOLUTE
QUANTIFICATION
ABSOLUTE
QUANTIFICATION
ABSOLUTE
QUANTIFICATION
ABSOLUTE
QUANTIFICATION

A difference of 0,1 in term of efficiency determines a

five-fold difference in the quantification after 30
cycles
ABSOLUTE
QUANTIFICATION
ACCURACY AND
TRUENESSACCURACY
The degree of agreement between a single value
obtained upon a test result and the accepted
reference value

TRUENESS
The degree of agreement between the mean
value obtained upon a series of test results and
the accepted reference value

The difference is known as BIASabs

 PRECISI
ON
A measurement that is precise
means that it agrees with other
measures of the same thing
Precision: degree of agreement among test results on replicates of the same
sample carried out in given conditions of repeatability or reproducibility (rif. ISO
5725-1).
 PRECISI
ON
Repeatability

For repeatability to be established, the following conditions

must be in place: the same location; the same
measurement procedure; the same observer; the same
measuring instrument, used under the same conditions;
and repetition over a short period of time. 
 PRECISI
ON
Reproducibility

Reproducibility, on the other hand, refers to the degree of

agreement between the results of experiments conducted
by different individuals, at different locations, with different
instruments. Put simply, it measures our ability to replicate
the findings of others. Through their extensive research, 
controlled inter-laboratory test programs are able to
determine reproducibility.   
PRECISI
ON
ACCURACY AND
PRECISION
ACCURACY AND
PRECISION
Accuracy is the degree of closeness to true value.

Precision is the degree to which an instrument or

process will repeat the same value.

In other words, accuracy is the degree of veracity

(exactness) while precision is the degree of
reproducibility.
ROBUSTN
ESS
ROBUSTN
ESS
ROBUSTN
ESS
ROBUSTN
ESS
EFFICIENCY (OR
EFFICACY)
LINEAR
ITY
LINEAR
ITY
 LINEAR
ITYand quantification
Detection
of HCV genotypes