Transcript LiMA overview

Overview of the presentation
1.
2.
3.
The LiMA technology
Sensitivity for ligase detection
Sensitivity for bacterial detection
The LiMA technology -1
Concept
• NAD-dependent DNA ligase is a universal bacterial
enzyme not found in mammalian systems
• Inherent amplification cascade. A single molecule of ligase
ligates many DNA substrate molecules which are then
amplified by PCR leading to very high sensitivity
• Pan-specific for bacteria. All bacteria contain NADdependent DNA ligase
LiMA – advantages compared to direct
detection of bacterial genomes by PCR
• LiMA is generic – all bacteria tested contain NAD-dependent
DNA ligase. It is difficult to ensure that direct PCR is generic.
• LiMA is more sensitive than direct PCR. LiMA involves lysis of
the bacilli and release of many ligase molecules which amplify
the target prior to sampling for PCR. This increases the chance
of detecting low numbers of organisms and diminishes any
inhibition of the PCR. In contrast, direct PCR can only amplify a
portion of the extracted material which may or may not contain
enough target to be detected (a single organism and its genome
can only be detected if it ends up in the PCR).
• LiMA can be used as a marker of viable bacteria – the ligase
disappears with loss of viability. Direct PCR will detect the
genome of dead bacilli.
The LiMA technology -2
Schematic
DNA substrate
5’
In the presence of
bacterial ligase
and NAD
DNA product
DNA product is
detected by PCR
PCR product
3’
3’ P 5’
3’
5’
Sensitivity of LiMA for NAD-dependent DNA ligase
detection
Serial dilutions of purified E.coli DNA ligase were tested by LiMA.
of free
E.coli
Ligase
The cycle atDetection
which the PCR
was
positive
was then plotted against
the micro Units of ligase used. Each dilution was tested in triplicate.
Ct values
40
0
35
0.01
30
0.1
1
25
10
20
100
15
0.01 0
1000
0.1
1
10
100
1000
micro UNITS of NEB Ligase
The LiMA could detect as few as 0.1 micro Units of ligase which is equivalent to
about 4000 molecules
LiMA protocol for bacterial detection
1. Lysis of bacteria (5 min). We have optimised a
generic lysis method using a bead beater.
2. Ligation (30 min). The lysis occurs in ligation
buffer including DNA substrate so that ligation
begins immediately upon lysis.
3. PCR (45 min). A portion of the ligated mix is
placed directly into a real time PCR which
includes SYBR Green for monitoring.
Experiment to demonstrate the sensitivity of LiMA for
detection of bacteria – E.coli
Serial dilutions of E.coli bacteria estimated to contain 100, 10, 3.3 and 1.1 organisms were
prepared and tested in triplicate. The bacteria were also enumerated by plating out,
incubation and counting colonies to give the actual number of organisms used in the LiMA
test. The table below shows the estimated number of organisms used, the actual number
determined after colony growth and the PCR cycle at which each dilution was positive.
Estimated
number of
organism in
each test
100
10
3.3
1.1
0
Actual number of
organisms (counted
in triplicate with the
mean also shown)
Not counted
50, 46, 70
Mean = 55
23, 17, 32
Mean = 24
Not counted
0, 0 ,0
Mean = 0
PCR cycle at which
each triplicate LiMA
was positive with the
mean also shown
20.8, 21.8, 21.0
Mean = 21.2
24.9, 24.7, 25.5
Mean = 25
25.7, 26.1, 25.6
Mean = 25.8
27.9, 26.3, 26.9
Mean = 27.0
32.5, 32.4, 33.5
Mean = 32.8
From the table it can be seen that
the LiMA method was able to
detect low numbers of
organisms. The actual numbers
of organisms after plating and
counting was higher than initial
estimates but the highest dilution
of organisms could easily be
distinguished (mean PCR cycle
27) from the ‘no-organism’ control
(mean PCR cycle 32.8). From
these results it can be estimated
that the LiMA assay can detect
10 or fewer organisms.
Summary of LiMA
• High sensitivity for detection of ligase and bacteria (fewer than 10
bacteria)
• Uses DNA substrate that can be detected on NAT platforms. At the
moment on the PCR platform but other NAT platforms could be used.
• Simple and rapid generic protocol (2.5 hr)