Diagnostic multiplex PCR assay for the identification of the Liverpool,
Midlands 1 and Manchester CF epidemic strains of
Joanne L. Fothergilla, Anna L. Uptona, Tyrone L. Pittb, C. Anthony Harta,†, Craig Winstanleya,⁎
aDivision of Medical Microbiology, University of Liverpool, Daulby Street, Liverpool L69 3GA, United Kingdom
bLaboratory of HealthCare Associated Infection, Health Protection Agency, 61 Colindale Avenue, London, NW9 5HT, United Kingdom
Received 19 July 2007; received in revised form 10 September 2007; accepted 20 September 2007
Available online 29 October 2007
Individual PCR amplification tests have been developed for three UK CF epidemic strains, the Liverpool epidemic strain (LES), Midlands 1
and the Manchester epidemic strain (MES). We report a simple diagnostic multiplex PCR test that can be used to screen for all three of these
strains. To evaluate the test, we screened collections of LES, MES and Midlands 1 isolates, along with various CF and non-CF non-epidemic
Pseudomonas aeruginosa strains. The test was 100% sensitive and 100% specific in the identification of these UK CF epidemic strains.
© 2007 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.
Keywords: Cystic fibrosis; Pseudomonas aeruginosa; Epidemic strains; Diagnostics
Once established as chronic, infection of CF patients with
Pseudomonas aeruginosa is impossible to eradicate. The
original view was that CF patients acquired their own unique
strains of P. aeruginosa from the environment. However,
transmission of P. aeruginosa between CF has been reported in
Europe [1–4] and Australia [5,6]. In the UK, the three most
widely studied epidemic strains are the Liverpool epidemic
strain (LES) [1,7], the Midlands 1 strain [8,9], and the
Manchester epidemic strain (MES) . A survey of CF
centres in England and Wales  showed that the LES was the
most common clone amongst CF isolates, followed by the
Midlands 1 strain. Specifically, 11% of CF patients were
infected with the LES, which was found in 48% of the CF
centres in the UK. The same survey identified the Midlands 1
strain in 10% of isolates and in 29% of centres and the MES in
1% of isolates and in 10% of centres . The LES has also been
reported in Scotland  and is associated with increased
patient morbidity .
Individual PCR amplification tests have been described
previously for the LES [13,14], Midlands 1  and the MES
. In each case subtractive hybridisation was used to identify
genes or genomic islands specific to the individual strains. In
this study we report a combined multiplex PCR (M-PCR) assay
to monitor for the presence of all three of these UK CF epidemic
2. Materials and methods
The panel of P. aeruginosa strains and isolates used in this
study have been described previously [9,14] and consisted of 30
LES isolates, 12 Midlands 1 isolates, 12 MES isolates, and 13
genetically-distinct non-epidemic CF isolates and the control
strains clone C , PA01  and PA14 . All isolates had
Journal of Cystic Fibrosis 7 (2008) 258–261
☆This paper is dedicated to the memory of Professor Tony Hart, an
inspirational colleague who is much missed by all of us, and by many others
throughout the microbiological world.
⁎Corresponding author. Tel.: 44 151 7064388; fax: +44 151 706 5805.
E-mail address: C.Winstanley@liv.ac.uk (C. Winstanley).
1569-1993/$ - see front matter © 2007 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.
been typed using macrorestriction followed by pulsed-field gel
Oligonucleotide primers (Sigma-Genosys) used for PCR
amplification are listed in Table 1 along with the annealing
temperatures used. DNA for PCR amplification was prepared by
directly in 25 µl volumes containing 1.25 U of GoTaq DNA
polymerase (Promega), 1×TaqMaster (Helena Biosciences),
300 nM each primer, 1×Taq buffer, 2.5 mM MgCl2 and
100 mM nucleotides (dATP, dCTP, dGTP, dTTP). As an
alternative polymerase system Fermentas Taq polymerase
(Helena Biosciences) was also used with the relevant buffer
30 cycles consisting of 95 °C (1 min), annealing temperature
(1 min) and 72 °C (2 min) with an additional extension time at
Eppendorf MasterCycler or a multigene II (Labnet) thermal
cycler. For assay development and positive controls a mixture of
representative isolates of the LES, MES and Midlands 1 strains
We sought to include primer sets for the amplification of
several target amplicons within the same assay. In particular, the
assay was designed to target two LES markers, PS21  and
LESF9 , a Midlands 1 marker, MID1 , a marker for the
MES, MA15 , and a sample control. Combinations of
primers were tested using temperature gradient PCR amplifi-
cation in order to vary the annealing temperature. For the
control we tested seven primer sets for amplification of bacterial
16S rRNA genes or the oprL gene (Table 1). Although the four
epidemic strain marker primer sets were readily compatible over
a range of annealing temperatures (data not shown), we were
only able to obtain all of the relevant amplicons from a mixture
of the three epidemic strains when combining these primer sets
with the 341f/518r primer set  for the amplification of
bacterial 16S rRNA genes (Fig. 1A). An annealing temperature
of 53 °C was chosen for the M-PCR assay on the basis of the
temperature gradient profile (Fig. 1A).
We carried out a blind test of the M-PCR assay on a panel of
LES, MES, Midlands 1 and non-epidemic strain P. aeruginosa
isolates whereby the test was carried out without prior
knowledge of the true identity of any isolates. These isolates
had previously been identified using PFGE. Example assays are
shown in Fig. 1B One MES isolate yielded an additional non-
specific band that made interpretation more difficult but, other
than that, the test was 100% sensitive and specific when applied
to our panel. We selected a sub-panel of nine isolates
representing two isolates of each of the epidemic strains and
three non-epidemic strains. These were used to carry out M-
PCR assays using Fermentas Taq polymerase (Helena Bios-
ciences) instead of GoTaq (Promega), and using a Labnet
Multigene thermal cycler instead of the Eppendorf MasterCy-
cler. In each case the M-PCR assay results were clear, although
with GoTaq polymerase a faint additional band of b300 bp
could be seen with some isolates (Fig. 1C).
Since chronic infection with P. aeruginosa is associated with
greater morbidity and mortality in CF patients [19,20] it is
important to keep CF patients free from P. aeruginosa infection
for as long as possible. With increasing evidence for the
existence of transmissible strains the onus is on CF clinics to
Oligonucleotide primers used in this study
Name Oligonucleotide (5′ to 3′)TargetAmplicon size (bp)Reference
259J.L. Fothergill et al. / Journal of Cystic Fibrosis 7 (2008) 258–261
monitor their patients and adopt appropriate infection control
policies. A simple PCR test should prove to be a useful tool for
better informing clinicians charged with the responsibility for
making decisions about segregation in order to prevent
epidemic spread of P. aeruginosa strains amongst patients. In
addition, widespread use of simple PCR assays such as this
should enable the community to better monitor the spread of
The M-PCR assay reported in this study identifies three of
the main UK epidemic strains in a single test. Since reporting
the PS21 test for the LES  we  and others  have
noted the occurrence of occasional false positives. In order to try
and address this issue we now use two PCR markers for the LES
. Thus far we have not identified any non-LES
P. aeruginosa isolates testing positive for both markers, nor
have false PCR-positives for either the Midlands 1 or the MES
markers been reported. However, it is possible that such strains
exist. Thus, we would suggest that the M-PCR test is used for
initial screening purposes, but that any suspected positives
should be followed up using additional molecular tests such as
PFGE or random amplified polymorphic DNA analysis.
Control strains for each of the epidemic strains will be made
available on request by Tyrone Pitt (Tyrone.Pitt@hpa.org.uk) or
Craig Winstanley (C.Winstanley@liv.ac.uk). In addition, it is
important that regular molecular typing is carried out to identify
any emerging epidemic strains not covered by this test. It is
widely recognised that PCR diagnostic tests are quicker and
cheaper than typing using PFGE and others have sought to
quantify these benefits . Although the savings, both
financial and temporal, may vary depending on the expertise
and equipment available within individual laboratories, we
believe that the M-PCR assay represents a considerable
improvement on the necessity for PFGE analysis of all CF
This work was undertaken under the framework of the UK
CF Microbiology Consortium, an initiative funded by the Big
Lottery Fund in association with the Cystic Fibrosis Trust.
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Fig. 1. Agarose gel (2%) electrophoresis of M-PCR assay products.
(A) Temperature gradient PCR amplification using a control template of all
three epidemic strains with varying annealing temperatures of, for lanes 1–10
respectively, 45.3, 46.4, 48.2, 50.4, 53.0, 55.8, 58.5, 61.0, 63.1 and 64.7 °C.
(B) Example PCR amplicons from a blind test of isolates. Isolates were
identified as LES (lanes 4 and 6), MES (lanes 1 and 3), Midlands 1 (lane 7) or
non-epidemic (unique) P. aeruginosa (lanes 2 and 5). (C) M-PCR assay using
GoTaq polymerase (Promega). MID; Midlands 1. M; 1 kb-plus size marker
(Invitrogen). C; Control mixture of LES, MES and Midlands 1.
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