Overview of the presentation

1. The LiMA technology

2. Sensitivity for ligase detection

3. Sensitivity for bacterial detection

The LiMA technology -1Concept

• 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 NAD-dependent 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.

3’ 3’P 5’ 5’5’ 3’

In the presence of bacterial ligase and NAD

DNA product is detected by PCR

DNA substrate

DNA product

PCR product

The LiMA technology -2

Schematic

Sensitivity of LiMA for NAD-dependent DNA ligase detection

Serial dilutions of purified E.coli DNA ligase were tested by LiMA. The cycle at which the PCR was positive was then plotted against the micro Units of ligase used. Each dilution was tested in triplicate.

The LiMA could detect as few as 0.1 micro Units of ligase which is equivalent to about 4000 molecules

0

Detection of free E.coli Ligase

15

20

25

30

35

40

0.01 0.1 1 10 100 1000

micro UNITS of NEB Ligase

Ct

valu

es

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0.01

0.1

1

10

100

1000

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.

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.

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.

Estimated number of

organism in each test

Actual number of organisms (counted in triplicate with the mean also shown)

PCR cycle at which each triplicate LiMA was positive with the

mean also shown

100 Not counted 20.8, 21.8, 21.0 Mean = 21.2

10 50, 46, 70 Mean = 55

24.9, 24.7, 25.5 Mean = 25

3.3 23, 17, 32 Mean = 24

25.7, 26.1, 25.6 Mean = 25.8

1.1 Not counted 27.9, 26.3, 26.9 Mean = 27.0

0 0, 0 ,0 Mean = 0

32.5, 32.4, 33.5 Mean = 32.8

Experiment to demonstrate the sensitivity of LiMA for detection of bacteria – E.coli

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)