Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Design of a single-tube, endpoint, linear-after-theexponential-PCR assay for 17 pathogens associated with
sepsis
L.M. Rice1, A.H. Reis Jr1, B. Ronish1, R.K. Carver-Brown1, J.W. Czajka2, N. Gentile3, G. Kost3 and
L.J. Wangh1
1 Department of Biology, Brandeis University, Waltham, MA, USA
2 Smiths Detection Diagnostics, Edgewood, MD, USA
3 Point-of-Care Technologies Center (National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health), Point-of-Care
Testing Center for Teaching and Research, Pathology and Laboratory Medicine, School of Medicine, University of California, Davis, CA, USA
Keywords
endpoint, fluorescent contours, fluorescent
signatures, linear-after-the-exponential-PCR,
septicaemia, sequence-specific low
temperature probes.
Correspondence
Lawrence J. Wangh, Department of Biology,
Brandeis University, Waltham, MA 024532728, USA. E-mail: [email protected]
2013/1001: received 1 June 2012, revised 2
October 2012 and accepted 30 October 2012
doi:10.1111/jam.12061
Abstract
Aims: The goal of this study was to construct a single-tube multiplex
molecular diagnostic assay using linear-after-the-exponential (LATE)-PCR for
the detection of 17 microbial pathogens commonly associated with
septicaemia.
Methods and Results: The assay described here detects 17 pathogens
associated with sepsis via amplification and analysis of gene-specific sequences.
The pathogens and their targeted genes were: Klebsiella spp. (phoE);
Acinetobacter baumannii (gyrB); Staphylococcus aureus (spa); Enterobacter spp.
(thdF); Pseudomonas aeruginosa (toxA); coagulase-negative staphylococci (tuf),
Enterococcus spp. (tuf); Candida spp. (P450). A sequence from an unidentified
gene in Lactococcus lactis, served as a positive control for assay function. LATEPCR was used to generate single-stranded amplicons that were analysed at
endpoint over a wide range of temperatures in four fluorescent colours. Each
target was detected by its pattern of hybridization to a sequence-specific
low-temperature fluorescent probe derived from molecular beacons.
Conclusions: All 17 microbial targets were detected in samples containing low
numbers of pathogen genomes in the presence of high levels of human
genomic DNA.
Significance and Impact of the Study: This assay used new technology to
achieve an advance in the field of molecular diagnostics: a single-tube assay for
detection of pathogens commonly responsible for septicaemia.
Introduction
Linear-after-the-exponential polymerase chain reaction
(LATE-PCR) is an advanced form of nonsymmetric PCR
in which amplification of double-stranded DNA is followed by efficient production of single-stranded amplicons. LATE-PCR also allows for a high degree of
multiplexing followed by endpoint detection by hybridization of low-temperature fluorescent probes in multiple
colours over a wide range of temperatures (Sanchez et al.
2004, 2006; Hartshorn et al. 2005a,b, 2007; Pierce et al.
2005; Salk et al. 2006; Rice et al. 2007). This highly
multiplexed single-tube approach is not achievable using
conventional symmetric PCR which only generates
double-stranded amplicons.
We have previously described relatively simple LATEPCR assays for foot-and-mouth disease virus (Pierce
et al. 2009), African swine fever virus (Ronish et al.
2010) and mitochondrial DNA mutational load (Osborne
et al. 2009). The highly multiplexed assay described here
can amplify and detect any one of 17 possible bacterial
and fungal targets commonly associated with septicaemia,
a condition that must be diagnosed in a matter of hours
because of its high rate of mortality (Balk et al. 2001).
Journal of Applied Microbiology 114, 457--469 © 2012 The Society for Applied Microbiology
457
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L.M. Rice et al.
The assay was constructed by first identifying specific
gene sequences unique to each bacterium or fungus. The
limiting and excess primers and a fluorescent probe for
each of these targets were then combined stepwise to
build the multiplex assay. The complete assay described
here has undergone preliminary evaluation at the University of California at Davis, School of Medicine and the
results of that study are being published as an accompanying paper (Gentile et al. 2012).
Materials and methods
Configuration of the sepsis gene specific LATE-PCR
multiplex assay
The scheme shown in Table 1 depicts the location of each
of the pathogens in a two dimensional detection space
comprised of fluorescent colour and temperature. Lowmelting temperature (Tm) molecular beacons, 64–43˚C, in
four fluorescent colours were used to detect their corresponding targets with high specificity. Subdivision of the
colour/temperature space in this manner made it possible
to detect 17 different pathogens plus an internal control.
Table 2 shows the pathogens by dye channel, as well as
the target genes and the GenBank accession ID numbers
from which the primers and probes were designed.
Design features of LATE-PCR multiplex assays
LATE-PCR is an advanced form of nonsymmetric PCR.
Each monoplex reaction utilizes a limiting primer (L)
and an excess primer (X) whose initial Tm fit the formula
Tm0X 0 (Sanchez et al. 2004). Primers of this
Tm0L
design efficiently generate double-stranded amplicons
until the limiting primer runs out and the reaction
switches to linear amplification of a single-stranded
amplicon driven by extension of the excess primer. The
accumulated single-stranded targets are measured at endpoint, by hybridization to low-temperature sequencespecific fluorescent probes.
Construction of a highly multiplexed assay for sepsis
began with bioinformatic identification of specific primer
pairs that would amplify gene-specific sequences character-
istic of a particular organism. Each gene sequence was scrutinized using Basic Local Alignment Search Tools (BLAST)
and was then compared with all other sequences in the
NCBI Genbank. The chosen sequences were then further
analysed to identify relatively conserved organism-specific
targets. Each of these sequences, in turn, was downloaded
into VISUAL OMP (VOMP) software (DNASoftware, Inc.,
Ann Arbor, MI, USA) for primer and probe design.
LATE-PCR primers and probes were designed with
multiplexing in mind using the following criteria. All
limiting primers were picked to have average concentration-dependent Tm’s of 711°C (range 736–680°C),
and all excess primers of 682°C (range 714–660°C).
In addition, the Tm of each excess primer was designed
to have a lower Tm than the Tm of its paired limiting
primer. The design process also circumvented possible
primer-primer or primer-probe interactions. Each pair
of prospective primers and its corresponding probe
sequences were then run through another BLAST search
to confirm that they had the appropriate homology to
identify the target sequences, and were also sufficiently
divergent from related targets to prevent amplification
or detection of the wrong target. Collectively these precautions greatly reduce the chances of false positives.
Table 3 describes the final primers, probes and amplicons in terms of their sequences and in silico Tm’s as
determined using VOMP software. Conserved gene
regions were selected on either the species level, or across
several species, as shown in Table 3. Some of the chosen
pathogen targets had large numbers of strain sequences
to examine which aided in design. A few of the pathogens, namely Enterobacter cloacae, had very little sequence
data available, so the amplicon was designed from the
single available sequence. Also, the dye channel and temperature space were chosen to detect each of the specific
products at endpoint with a low Tm mismatch tolerant
probe. Primers and probes were designed in an effort to
conserve, and make efficient use of the available fluorescent colour and temperature space leading to all species
being detected.
Target-specific sets of primers and probes were first
tested in monoplex assays using genomic DNA of the
relevant pathogen as the target. All primer pairs and all
Table 1 The linear-after-the-exponential-PCR multiplex assay configuration
Temperature, °C
458
FAM
Cal Orange
Cal Red
Quasar
Klebsiella pneumoniae
Acinetobacter baumannii
Klebsiella oxytoca
Lactococcus lactis
Staphylococcus aureus
Enterobacter aerogenes
Enterobacter cloacae
Pseudomonas aeruginosa
Staphylococcus haemolyticus
Staphylococcus saprophyticus
Staphylococcus epidermidis
Staphylococcus hominis
Candida albicans
Candida glabrata
Enterococcus faecalis
Enterococcus faecium
Candida parapsilosis
Candida tropicalis
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LATE-PCR multiplex assay for sepsis
Table 2 Reference sequences for primer probe design in linear-after-the-exponential-PCR multiplex assay
Channel
Target
Gene
Accession ID
Acquisition
FAM
Klebsiella pneumoniae
Acinetobacter baumannii
Klebsiella oxytoca
Lactococcus lactis
Staphylococcus aureus
Enterobacter aerogenes
Enterobacter cloacae
Pseudomonas aeruginosa
Staphylococcus haemolyticus
Staphylococcus saprophyticus
Staphylococcus epidermidis
Staphylococcus hominis
Candida albicans
Candida glabrata
Enterococcus faecalis
Enterococcus faecium
Candida parapsilosis
Candida tropicalis
phoE
gyrB
phoE
Unknown
spa
thdF
thdF
toxA
tuf
tuf
tuf
tuf
P450
P450
tuf
tuf
P450
P450
M28295
NC_011595
X68022
NC_008527
AM076292
EU569333
EU569339
NC_008463
AF298801
AF298804
AF298800
AF298802
AF153846
AY942647
AE016830
NZ_ACHL01000063
GQ302972
AY942643
ATCC: BAA-1706D -5
ATCC: 17978D-5
ATCC: 700324D-5
Aliquot Edgewood
Aliquot PHRI
ATCC: 15038D-5
ATCC: 13047D-5
ATCC: 17933D-5
ATCC: 29970D-5
ATCC: 15305D-5
ATCC: 12228D-5
ATCC: 700236D-5
ATCC: 14053D-5
ATCC: 15545D-5
ATCC: 700802D-5
ATCC: BAA-472D-5
ATCC: 22019D-5
ATCC: 750D-5
CalOrg
CalRed
Quasar
probes were combined into a multiplex assay which was
tested separately against the genomic DNA of each pathogen. All such reactions were carried out in triplicate along
with many replicates of no-template-controls (NTC).
This iterative process allows for optimization of all
components in such a multiplex reaction. Reaction mixtures that were efficient using high number of genomic
copies were then tested even more rigorously by 10-fold
serial dilution of the genomic DNA from 105 to 10 2
copies per reaction. Only those reactions that have very
low levels of nonspecific hybridization are reliable at
high dilution. Genomic DNA used to determine the
limit of detection for each pathogen was first quantified
via real-time monoplex LATE-PCR analysis with SYBR
Green.
Assay composition
Every experiment had a final volume of 25 ll and contained the same reaction component, mixture comprised
of: 19 PCR buffer (cat. no. 10966-034; Invitrogen, Carlsbad, CA, USA), 3 mmol l 1 MgCl2 (cat. no. 10966-034;
Invitrogen), 250 nmol l 1 dNTPs (Invitrogen), PCR
Grade Water (cat.no. BP2819-1; Fisher Scientific),
100 nmol l 1 of each probe (Biosearch Technologies,
Novato, CA, USA), 50 nmol l 1 of each limiting primer
(Sigma-Aldrich, St Louis, MO, USA) and 1000 nmol l 1
of each excess primer (Sigma-Aldrich). All reactions contained 125 units of Platinum Taq DNA polymerase (cat.
no. 10966-034; Invitrogen) and 1 ll of target genomic
DNA. Target genomic DNA (Table 2) was received from
American Type Culture Collection (ATCC, Manassas,
VA, USA) as a dry reagent (5 µg) and was suspended in
500 ll of 10 mmol l 1 Tris-Cl pH 83 (Sigma-Aldrich)
for a final genomic DNA concentration 106 genomes per
µl or as previously characterized suspended purified
genomic DNA from University of California Davis,
Smiths Detection Diagnostics or Public Health Research
Institute (PHRI). SYBR Green dilution series were used
to verify the starting genomic DNA copy number for all
pathogens (Dhanasekaran et al. 2010). All multiplex mixture and limit of detection reactions contained
10 000 copies of human genomic DNA (cat. no. G304A;
Promega, Madison, WI, USA) which was included to
verify the robustness and specificity of the reaction.
LATE-PCR protocol and thermal amplification
parameters
Each reaction was run in triplicate in either a Bio-Rad IQ5
Multicolor Real-Time Detection System (Bio-Rad Laboratories, Hercules, CA, USA) or in the Stratagene Mx3005P
(Agilent Technologies, Santa Clara, CA, USA). No significant endpoint differences were observed for the two
machines. Three NTCs were also run in parallel in every
experiment. An initial denaturation step at 95°C for 3 min
was followed by 50 amplification cycles of 95°C for 10 s,
65°C for 15 s and 72°C for 30 s. Next, the temperature was
dropped from 72 to 25°C. Beginning at 25°C, the temperature was increased in 56 cycles of 30-s steps and 1°C increments to 80°C. Empirical observation suggests that this
down-up process improves the homogeneity of molecules
in the reaction (data not shown). Once the molecules have
reached this level the reproducibility of the data is
enhanced. Finally, endpoint data collection was carried out
by decreasing the temperature, annealing in 1°C
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459
LATE-PCR multiplex assay for sepsis
L.M. Rice et al.
Table 3 Primer and probe sequences in linear-after-the-exponential-PCR multiplex assay
Name
Sequence 5′–3′
Bases number
Tm °C
Klebsiella pneumoniae amplicon
GCTTTGTGGCTTCAACGGCGACGCAGGCAGCGGAAGTTTA
TAATAAGAACGCGAACAAGCTGGATGTGTACGGCAAGA
TCAAAGCCATGC ACTACTTCAGCGA
TCGCTGAAGTAGTGCATGGCTTTGATC
AAAGCTTTGTGGCTTCAACGGCGACG
BHQ-1-TAAAGAACGCGAACAAGCTGGTA- FAM
GCATTGCAATGGAACGATAGTTACCAAGAAAATGTTCGCTG
TTTCACAAACAACATTCCACAAAAAGATGGTGGTACGCAC
TTAGCAGGTTTCCGCGCAGCTTTAACACGTGGCTTAAACC
AGTATCT
GCATTGCAATGGAACGATAGTTAC
AGATACTGGTTTAAGCCACGTGTTAAAGCTGCG
BHQ1 – TGTGTGAAACAGCGAACATTCA – FAM
GATGATGGGTCTTATTGCTTCTTCGGCTACCCAGGCGGCAGA
AGTTTATAATAAAAACGGCAATAAACTGGACGTCTATGGCA
AAGTCAAA GCGATGCACTATATGAGCCT
AGGCTCATATAGTGCATCGTTTTGACT
GATGATGGGTCTTATTGCTTCTTCGGCTACC
BHQ1 –TTCGGCAATAAACTGGACGTCTATAA – FAM
TAATCATTATTCCTCAAGAAGAGATACAATCGGTCACTTTTAA
GAAAGGTTTACTTGCTTATAAAATGGTTGTGACTACTAAAG
ATAACGAA GTTCCTGATTTTAG
TAATCATTATTCCTCAAGAAGAGATACAATCGGTCA
CTAAAATCAGGAACTTCGTTATCTTTAGTAGTCACAACCA
BHQ 1 – ATAAACCTTTCTTAAAAT – FAM
TGAACATGCCTAACTTGAACGAAGAAC
AACGCAATGGTTTCATCCAAAGCTTAAAAGATGACCCA
AGTCAAAGTGCTAACCTTTTAGCAGAAGCTAAAAAGTT
AAATGAATCTCAAGCACCGAAAGC
TGAATATGCCTAACTTGAACGAAGAACAACG
GCTTTCGGTGCTTGAGATTCATTTAACTTTTTAGCTTCTG
Cal Orange – AATGGGTCATCTTTTAAGCTTTGGTT – BHQ1
GTGGAAGCGCTCACTCACCTGCGCATCTACGTGGAAGCGGC
GATTGATTTCCCGGATGAAGAAATTGATTTCCTCTCCGATGG
TAAAATTGAAGC
GCTTCAATTTTACCATCGGAGAGGAA
GTGGAAGCGCTTACTCACCTGCGCATCT
Cal Orange – TCTTCCCGGATGAAGAAATGA – BHQ1
TCTTGTGGAAGCACTTACTCACCTCAGGATCTACGTCGAAG
CAGCGATTGACTTCCCGGATGAAGAAATCGACTTTCTCTC
TGACGGTAAAA TTGAAGC
GCTTCAATTTTACCGTCAGAGAGAAA
TCTTGTGGAAGCACTTACTCACCTCAGGATCT
TGAACTGGCTGGTACCGATCGGCCACGAGAAGCCCTCGA
ACATCAAGGTGTTCATCCACGAACTGAACGCCGGTAACC
AGCTCAG
TGAACTGGCTGGTATCGATCGG
CTGAGCTGGTTACCGGCGTTCAGTTC
Cal Red – AAGGATGAACACCTTGATGTTCGATT – BHQ2
103
790
27
26
23
128
695
713
644
830
24
33
22
111
663
720
581
857
27
31
26
106
689
718
643
780
36
40
18
127
684
697
439
852
31
40
26
95
714
736
624
843
26
28
21
99
682
720
556
824
26
32
85
659
710
878
22
26
26
699
712
631
Kl. pneumoniae excess primer
Kl. pneumoniae limiting primer
Kl. pneumoniae probe
Acinetobacter baumannii amplicon
Ac. baumannii excess primer
Ac. baumannii limiting primer
Ac. baumannii probe
Klebsiella oxytoca amplicon
Kl. oxytoca excess primer
Kl. oxytoca limiting primer
Kl. oxytoca probe
Lactococcus lactis amplicon
L. lactis excess primer
L. lactis limiting primer
L. lactis probe
Staphylococcus aureus amplicon
Staph. aureus excess primer
Staph. aureus limiting primer
Staph. aureus probe
Enterobacter aerogenes amplicon
Ent. aerogenes excess primer
Ent. aerogenes limiting primer
Enterobacter probe
Enterobacter cloacae amplicon
Ent. cloacae excess primer
Ent. cloacae limiting primer
Pseudomonas aeruginosa amplicon
Ps. aeruginosa excess primer
Ps. aeruginosa limiting primer
Ps. aeruginosa probe
(Continued )
460
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L.M. Rice et al.
LATE-PCR multiplex assay for sepsis
Table 3 (Continued )
Name
Sequence 5′–3′
Bases number
Tm °C
Staphylococcus haemolyticus amplicon
GCCGTGTTGAACGTGGGCAAATCAAAGTTGGTGAAGAAG
TTGAAATCATTGGTATCCATGACACTTCTAAAACAACTGT
TACTGGTGTAGAAATGTTCCGTAAATTATTAGACTACGCTG
AAGCTGGTGACAACATCGGTGCATTATTACGTGGTGTTGCTC
GTGAAGACGTACAACGTGGTCAAGTATTAGCGCCGTGTTGA
ACGTGGGCAAATCAAAGTTGGTGAAGAAGTTGAAATCATTG
GTATCCATGACACT
GCCGTGTTGAACGTGGTCAAATCAAAGTCGGTGAAGAAATCG
AAATCATCGGTATGCAAGAAGAATCCAAAACAACTGTTACTG
GTGTAGAAATGTTCCGTAAATTATTAGACTACGCTGAAGCTGG
TGACAACATTGGTGCATTATTACGTGGTGTTTCACGTGATGATGT
ACAACGTGGTCAAGTTTTAGCGCCGTGTTGAACGTGGTCAAAT
CAAAGTCGGTGAAGAAATCGAAATCATCGGTATGCAAGAAGAA
GCCGTGTTGAACGTGGTCAAATCAAAGTTGGTGAAGAAGTTGAAA
TCATCGGTATGCACGAAACTTCTAAAACAACTGTTACTGGTGTAG
AAATGTTCCGTAAATTATTAGACTACGCTGAAGCTGGTGACAACA
TCGGTGCTTTATTACGTGGTGTTGCACGTGAAGACGTACAACGTG
GTCAAGTATTAGCGCCGTGTTGAACGTGGTCAAATCAAAGTTGGT
GAAGAAGTTGAAATCATCGGTATGCACGAAACT
GCCGTGTTGAACGTGGTCAAATCAAAGTTGGTGAAGAAGTTGAAA
TTATTGGTATCAAAGAAACTTCTAAAACAACTGTTACTGGTGTAGA
AATGTTCCGTAAATTATTAGACTACGCTGAAGCTGGTGACAACATC
GGTGCTTTATTACGTGGTGTTGCTCGTGAAGATGTACAACGTGGTC
AAGTATTAGCGCCGTGTTGAACGTGGTCAAATCAAAGTTGGTGAA
GAAGTTGAAATTATTGGTATCAAAGAAACT
TTCATCTTTAGATAAAACGTATACGTCTGCTTTGAATTTTGTGT
GTCGGGTTGAACGTGGTCAAATCAAAGTTGGTGAAGA
CalRed- AACTGTTACTGGTGTAGAATT-BHQ2
ATCAGAATACGATTTCCCAGGCGATGATGTTCCAGTTATCGCAGGTTC
TGCTTTGAAAGCTTTAGAAGGCGACGAGTCTTATGAAGAAAAAATC
TTAGAATTAATGGCTGCAGTTGACGAATATATCCCAACTCCAGAAC
GTGATACTGACAAACCATTCATGATGCCAGTCGAAGACGTATTCTC
AATCACTATCAGAATACGATTTCCCAGGCGATGATGTTCCAGTTAT
CGCAGGTTCTGCTTTGAAAGCTT
AACAGAATACGAATTCCCTGGTGACGATGTTCCTGTAGTTGCTGGTTC
AGCTTTGAAAGCTCTAGAAGGCGACGCTTCATACGAAGAAAAA AT
TCTTGAATTGATGGCTGCAGTTGACGAATACATCCCAACTCCAGAAC
GTGACAACGACAAACCATTCATGATGCCAGTTGAAGACGTGT TCTC
AATCACTGGACGTGGTACTGTTGCTACAGGTCGTGTGGAACGTGGAC
AAGTTCGCGTTGGTGACGAAGTTG
ATCAGAATACGAGTTCCCTGGTGATGATGTTCC
CCACTTCGTCACCAACGCGAACTTCTCCACGTTC
Quasar 670 – AAGCATCATGAATGGTTTGTT – BHQ2
ACCTTTACCTCATTATTGGAGACGTGATGCTGCTCAAAAGAAAATCTCT
GCTACTTATATGAAAGAAATTAAACTGAGAAGAGAACGTGGTGATAT
TGATCCAAATCGTGATTTAATTGATTCCTTATTGATTCATTCAACTTATA
AAGATGGTGTGAAAATGACTGATCAAGAAATTGC
ACCATTACCTCATTATTGGAAACGTGATGCTGCGCAACAAAAGATTTCT
GAAACGTATATGACAGAGATTGCTAGAAGAAGAGAGACGGGTGACA
TTGATGAAAATCGTGATTTAATCGATTCTTTATTGGTAAACTCTACATAC
AAAGATGGTGTTAAAATGACTGATCAGGAAATTGC
260
877
260
874
260
873
260
866
44
37
21
257
66
69
59
866
257
877
33
34
21
180
668
717
555
750
180
760
Staphylococcus saprophyticus amplicon
Staphylococcus epidermidis amplicon
Staphylococcus hominis amplicon
Coag Negative excess primer
Coag Negative limiting primer
Coag Negative probe
Enterococcus faecalis amplicon
Enterococcus faecium amplicon
Enterococcus excess primer
Enterococcus limiting primer
Enterococcus probe
Candida albicans amplicon
Candida parapsilosis amplicon
(Continued )
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LATE-PCR multiplex assay for sepsis
L.M. Rice et al.
Table 3 (Continued )
Name
Sequence 5′–3′
Bases number
Tm °C
Candida tropicalis amplicon
ACCATTACCTCATTACTGGAGACGTGACGCTGCTCAAAGAAAGATATC
TGCTCATTACATGAAGGAAATCAAGAGAAGAAGAGAAAGCGGTGAT
ATTGATCCAAAGAGAGATTTGATTGATTCCTTGTTGGTTAACTCTACTTA
TAAAGATGGTGTTAAAATGACTGATCAAGAAATTGC
GCAATTTCTTGATCAGTCATTTTTACACCATCTT
ACCATTACCTCATTATTGGAGACGTGATGCTGC
Quasar 670 – ATGTGATATTGATCCAAATCGTGATTTAATAT-BHQ2
ATGCCCAACAAGCTATCTCTGGTACTTACATGTCCTTGATTAAGGAAAG
ACGTGAGAAGAACGATATCCAAAACCGTGACTTGATTGATGAATTG
ATGAAGAACTCCACTTACAAGGATGGTACTAAGATGACCGACCAAGA
AATTGCCAACCTATTGATTGGTGTCTTGATGGGTGGTCAACATACTTCCG
CGGATGTTGCAGGGGAAGTATGTTGACCACCCA
ATGCCCAACAAGCTATCTCTGGTACTTACATGT
Quasar 670-AAACAAGGATGGTACTAGGATGACCGTT-BHQ2
180
760
34
33
32
204
670
680
63/50
881
33
33
28
701
722
62
Candida APT excess primer
CandidaAPT limiting primer
Candida APT probe
Candida glabrata amplicon
C. glabrata excess primer
C. glabrata limting primer
C. glabrata probe
Coag Negative, coagulase-negative staphylococcus; Candida APT, C. albicans, C. parapsilosis, and C. tropicalis.
decrements at 30 s intervals for 56 steps between 80 and
25°C. Fluorescence was measured in the FAM, CalOrg560,
CalRed610 and Quasar670 channels at each anneal step.
Data analysis
The fluorescent contours were generated as follows: The
raw fluorescent endpoint data for each temperature step
and each dye channel collected on either the Bio-Rad
IQ5 or the Stratagene Mx3005P were exported to Microsoft Excel 2007. Each set of data was normalized by
dividing all values by the value of the signal at 70°C in
that data set. This was because no probe was bound to
any target at this temperature and the fluorescence intensity in each colour was at the background level for all
probes of that colour. Next, each data set was corrected
for background temperature-dependent levels of fluorescence due to unbound probes in that colour. This was
accomplished by subtracting the background temperature-dependent level of fluorescence of an average NTC
sample from each corresponding experimental sample in
that colour. At this point all positive fluorescent signals
above background for each pathogen in their respective
colour were re-scaled by dividing all fluorescent values in
a data set by the highest fluorescent value in that data
set. The resulting fluorescent values describe a temperature-dependent fluorescent contour in relative fluorescent
unit (RFU) values on a scale of 0-to-1. The fluorescent
signatures illustrated in this article are the first derivatives
of the raw fluorescent data collected by either the BioRad IQ5 or the Stratagene Mx3005P, and were generated
using the algorithms built into each machine.
462
Results
The results presented here describe the design of a highly
multiplexed LATE-PCR single-tube assay for detection of
17 microbial pathogens commonly associated with septicaemia. The complete LATE-PCR sepsis multiplex assay
was comprised of 24 primers and 11 probes distributed
into four fluorescent colours as shown in Table 1. The
success of the assay rests on the bioinformatic analysis of
the chosen DNA sequences which were both target specific and compatible with properties of LATE-PCR limiting and excess primers.
Figure 1 displays the results of the fluorescent contours
and their corresponding fluorescent signatures for each of
the genomic DNA targets analysed one-by-one in triplicate in a master mix of all primers and probes. The data
in Fig. 1 were generated using 106 copies of each genome.
The results established that each target pathogen had its
own unique fluorescent pattern, except for a group of
four coagulase-negative staphylococci (CoNS) (Fig. 1
Panel c1) that had a unique fluorescent signature, but
cannot be distinguished individually. These results were
expected and were a consequence of probe design. The
probe for the four CoNS was designed not to distinguish
on the species level. Consequently, as shown in Fig. c1,
the CoNS species (i.e., Staphylococcus haemolyticus, Staphylococcus saprophyticus, Staphylococcus epidermidis and
Staphylococcus hominis) have the same CalRed610 fluorescent signals and were detectable only as CoNS.
Some probes were designed to differentiate between
similar pathogens while still conserving the number of
probes used in the multiplex because of dye channel and
Journal of Applied Microbiology 114, 457--469 © 2012 The Society for Applied Microbiology
L.M. Rice et al.
temperature detection space restrictions. A single probe
can be designed for detection and differentiation of multiple pathogens by ensuring that each probe/target hybrid
has its own Tm. As shown in Fig. d1, the probe that
bound to Candida albicans at 63°C was the same probe
that bound to Candida tropicalis at 50°C (refer to
Table 3), and thus generated pathogen specific patterns.
A second Candida probe was designed for multiple pathogen detection for differentiation of two other Candida
pathogens to ensure that the probe to amplicon binding
produced distinctly specific contours through nucleotide
mismatching. Candida parapsilosis and C. tropicalis both
bound their detection probe at 50°C, however, the contours of their anneal curves looked distinctly different, as
shown in Fig. d1. At progressively lower temperatures the
C. parapsilosis signal dips below the C. tropicalis, thus
distinguishing the contours of these two species.
Limit of detection of genomic DNA samples
The limit of detection of each genomic DNA target is
critical to this assay’s clinically relevance, as in septicaemia the concentration of pathogens in a blood sample
varies from very low to very high depending on the
extent of infection. As shown in Table 4, the limit of
detection was dependent on several factors: the sample
preparation, the pathogen, the fluorescent dye channel,
the detection temperature, starting copy number, and the
amount of single-stranded DNA produced during LATEPCR amplification. A balance was achieved between the
efficiency of target amplification by optimization of primer design, and the number of cycles used for singlestranded amplicon accumulation. The more efficient the
primers or the larger the number of initial genomes, the
smaller the number of amplification cycles required to
reach a detectable concentration of a single-stranded
product. In this assay, the high level of primer specificity
and low level of nonspecific interactions allowed for
detection of small numbers of initial genomes. Positive
detection of a particular pathogen was made when all
three replicates were above the NTC background adjusted
for each fluorescent colour. Typically, the signal to background ratio in the FAM dye channel was lower in comparison with the other dye channels utilized in this assay.
The CalRed610 and Quasar670 dye channels produced
higher fluorescent signals relative to background fluorescence. Lower temperature probes were more affected by
background fluorescent levels because of increasing secondary structure of the target at lower temperatures.
When background fluorescent levels were high, the same
low temperature probes do not generate a high enough
signal for the lower copy numbers to be distinguishable
from the background fluorescence.
LATE-PCR multiplex assay for sepsis
The genomic DNA targets with the highest limits of
detection were Klebsiella oxytoca, Lactococcus lactis, Enterobacter aerogenes and Ent. cloacae with a limit of detection of 1000 copies. The Kl. oxytoca and the L. lactis,
annealing at the temperatures of 543 and 439°C, respectively, have a relatively high limit of detection because
they were in the FAM dye channel with their respective
probes expected to anneal at a lower temperature than
the Klebsiella pneumoniae at 644°C (limit of detection
10 copies) and Acinetobacter baumannii at 581°C (limit
of detection 100 copies). The use of four FAM probes in
the full multiplex of this assay detrimentally contributed
to background fluorescence. In the absence of target, the
unbound probes greatly increased the background making the lower annealing temperature probes (Kl. oxytoca
and the L. lactis) difficult to discern from background
fluorescence at low copy number. Acinetobacter baumannii (limit of detection 100 copies) was also affected by
background fluorescence in the FAM channel but not to
the same extent because its probe had a higher annealing
temperature relative to the Kl. oxytoca and L. lactis.
In the CalOrg560 dye channel, Ent. aerogenes and the
Ent. cloacae shared a single probe and had a limit of
detection of 1000 copies. The Enterobacter probe,
although perfectly matched to both amplicons, gave species specific probe contours because of the design of the
probe stem. The four nucleotides, which make up the
probe stem, were typically designed to bind to each other
and not the amplicon. The probe stem of the Enterobacter
probe was different for two reasons. The first reason was
that one half of the stem binds to the Ent. cloacae
sequence and not to the Ent. aerogenes sequence, providing for species specific probe contours. The second reason
was that the stem was composed of the nucleotides TC
and GA. Typically stem design for low temperature
molecular beacons uses only A’s and T’s to allow the
probe to readily open. The presence of the C/G nucleotide binding slowed the opening of the probe stem and
affected the probe’s binding efficiency, making it less efficient at the lower copy number, but still provided discrimination between the two targets. The other pathogen
that was detected in the CalOrg560 dye channel was
Staphylococcus aureus, which had a limit of detection of
10 copies with its probe binding at 62°C.
Another factor that induces a higher limit of detection
was the use of mismatched tolerant primers and probes
because they do not bind perfectly to the target and
could delay amplification. If a primer is mismatched but
the mismatch was not destabilizing, the effect on amplification delay can be minimized. All pathogens detected in
the CalRed610 dye channel had a limit of detection of
10 copies or less. Pseudomonas aeruginosa was detected
with a high-temperature probe (631°C). A slightly lower
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Figure 1 The detection of 17 pathogens associated with sepsis. Anneal curves (a1, b1, c1, d1) are fluorescent contours. All 1st derivative anneal
curves (a2, b2, c2, d2) are fluorescent signatures. (a1) FAM: Klebsiella pneumoniae – black dotted, Acinetobacter baumannii – black solid, Klebsiella oxytoca – black dashed and Lactococcus lactis – grey solid. (b1) CalOrg560: Staphylococcus aureus – black dashed, Enterobacter cloacae –
solid grey and Enterobacter aerogenes – solid black (c1) CalRed610: Pseudomonas aeruginosa – black dotted, coagulase-negative staphylococci;
including Staphylococcus hominis, Staphylococcus saprophyticus, Staphylococcus epidermidis and Staphylococcus haemolyticus – grey and black
solid and dashed. (d1) Quasar670: Candida albicans – black solid, Candida glabrata – black dotted, Enterococcus faecalis – black dashed and
Enterococcus faecium – grey dashed, Candida tropicalis – grey solid and Candida parapsilosis – black hashed.
464
Journal of Applied Microbiology 114, 457--469 © 2012 The Society for Applied Microbiology
L.M. Rice et al.
LATE-PCR multiplex assay for sepsis
Table 4 Limit of detection of genomic DNA targets by copy number in linear-after-the-exponential-PCR multiplex assay*
Temperature, °C
FAM
Cal Orange
Cal Red
Quasar
Klebsiella pneumoniae
10
Acinetobacter baumannii
100
Klebsiella oxytoca
1000
Lactococcus lactis
1000
Staphylococcus aureus
10
Enterobacter aerogenes
1000
Enterobacter cloacae
1000
Pseudomonas aeruginosa
10
Staphylococcus haemolyticus
10
Staphylococcus saprophyticus
10
Staphylococcus epidermidis
10
Staphylococcus hominis
10
Candida albicans
100
Candida glabrata
10
Enterococcus faecalis
100
Enterococcus faecium
100
Candida parapsilosis
100
Candida tropicalis
100
*Genomic DNA dilution series were previously quantified in monoplex using SYBR-Green (Applied Biosystems, Foster City, CA, USA).
temperature probe (59°C) detected pathogens as CoNS,
which included Staph. haemolyticus, Staph. saprophyticus,
Staph. epidermidis and Staph. hominis. It is significant to
note that there was a single primer pair that amplifies all
four CoNS. Each target was mismatched differently to
both the limiting and excess primers but all were perfectly matched to the probe. However, although both
primers were mismatched, the mismatches were not
destabilizing and the effect on amplification delay was
minimized and the limit of detection remained low.
Of the four fluorescent dyes used here, Quasar670 has
the most capacity for pathogen detection because its signal intensity over background is greatest, even at low
temperatures. For this reason, Quasar670 was chosen for
analysis of the largest number of pathogens (six), as compared to four in FAM, three in CalOrg560 and five in
CalRed610. The six pathogens analysed in Quasar can be
considered in three groups: (i) C. albicans, C. parapsilosis,
C. tropicalis; (ii) Candida glabrata and (iii) Enterococcus
faecalis, Enterococcus faecium. C. albicans, C. parapsilosis
and C. tropicalis were amplified by a single mismatch tolerant primer pair to the P450 gene target. As shown in
Table 3, the probe was perfectly matched to the C. albicans amplicon allowing for detection at 63°C. It was
intentionally designed to be mismatched to C. parapsilosis
and C. tropicals allowing for detection of all three species
down to a level of 100 copies. The C. glabrata P450 gene
target was not sufficiently conserved to be amplified by
the primers used for group 1 and was therefore amplified
by its own pair of primers and a probe which achieved a
limit of detection of 10 copies. The species in group 3,
Ent. faecalis and Ent. faecium were amplified with a set of
mismatch tolerant primers that amplified both species
and were distinguished using a perfectly matched probe
whose binding was impacted by an adjacent sequence
that had a distinctly different secondary structure in the
two species. This novel approach allowed the targets to
be distinguished all the way down to a limit of detection
of 100 copies.
Mixtures of genomic DNA samples
LATE-PCR primers detected low copy levels of a pathogen in a large background of other pathogens. This is
important in the clinical setting, in which mixtures of
pathogens can be present either through true polymicrobial infection or contamination of sample during acqusition. As shown in Table 1, pathogen detection was
divided into four different dye channels, such that some
pathogens were detected in a shared dye channel. For this
reason, LATE-PCR endpoint multiplex analysis of mixtures was tested between different dye channels and
within a single dye channel. In every multiplex experiment, when a single pathogen was present, only one dye
channel had a positive signal above background. When a
mixture of pathogens between dye channels was present,
two dye channels had separate positive signals above
background. This established that each pair of primers
functioned independently even though all the primers
and probes were present in the reaction mixture.
Mixtures of pathogens in this LATE-PCR multiplex
assay were carried out by preparation of genomic DNA
targets at a ratio of 99 : 1 using 105 and 103 copies of
the chosen genomes. All assays also contained a background of 104 copies of human genomic DNA to rigorously test primer and probe specificities. Figure 2
illustrates the normalized fluorescent contours for a representative set of results for the many possible combinations of pathogen mixtures across the four dye channels.
In this case, Staph. aureus was detected by virtue of
its signals in CalOrg560, which was present in each
two-pathogen mixture. The other dye channels FAM,
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L.M. Rice et al.
CalRed610 and Quasar670 were used for the detection of
the other variable pathogen in the two-pathogen mixture:
Kl. pneumoniae visible in FAM, Ps. aeruginosa visible in
CalRed610 and Ent. faecalis visible in Quasar670 (see
Table 1 for assay configuration).
In Fig. 2a, the FAM and CalOrg560 dye channels were
used to detect Kl. pneumoniae (FAM) at the 1% level in a
background of 99% Staph. aureus (CalOrg560) and 1%
Staph. aureus (CalOrg560) in a background of 99%
Kl. pneumoniae (FAM). In the CalRed610 dye channel
(Fig. 2b), Ps. aeruginosa (CalRed610) was detected at the
1% level in a background of 99% Staph. aureus
(CalOrg560) and 1% Staph. aureus (CalOrg560) in a
background of 99% Ps. aeruginosa (CalRed610). In the
Quasar670 dye channel (Fig. 2c), Ent. faecalis (Quasar670)
was detected at the 1% level in a background of 99%
Staph. aureus (CalOrg560) and 1% Staph. aureus
(CalOrg560) in a background of 99% Ent. faecalis
(Quasar670).
As a mixture of pathogens might also be present within
the same dye channel, tests were carried out to analyse
this possibility a well. In Fig. 3a, 1% Staph. aureus, was
mixed into a background of 99% Ent. aerogenes and both
were detected in the CalOrg560 dye channel. Positive
controls of 100% Staph. aureus and Ent. aerogenes were
run in parallel. In another experiment (Fig. 3b), 1%
Ps. aeruginosa was mixed in a background of 99% CoNS
(Staph. hominis) and was detected in the CalRed610
channel. Positive controls of 100% Ps. aeruginosa and
Staph. hominis were run in parralel. Referring back to
Table 1, other combinations of mixtures within a single
dye channel were possible; Figure 3 provides only two
representative examples. The differentiation of mixtures
in a single dye channel is only limited by the resolution
of specific fluorescent probes to specific pathogens.
Therefore, CoNS mixtures would not be resolvable nor
would mixtures of Ent. facalis and Ent. faecium, as in
these cases similar fluorescent contuors and fluorescent
signatures result.
Even more complex mixtures of pathogens were analysed by mixing three pathogens. Figure 4, through fluorescent signatures, illustrates the representative use of two
dye channels for the detection of mixtures of three pathogens. Pseudomonas aeruginosa and Staph. hominis which
were detected in the CalRed610 dye channel were mixed
with C. glabrata which was detected in the Quasar670
dye channel (see Table 1 for further clarification of assay
configuration). Equal mixtures of the three ATCC genomic DNA targets were made at both the 100 000 copy
level and the 1000 copy level to serve as positive controls.
Mixtures were also made so that individual genomic
DNA targets were at the 1000 copy level while the other
two remained at 100 000 copies. All mixtures were run
466
in parallel to allow comparison for detection of the mixture. As shown in Fig. 4, the mixture of Ps. aeruginosa
(CalRed610), Staph. hominis (CalRed610) and C. glabrata
(Quasar670) were detectable at the 1% level in a background of two other pathogens. Complex mixtures of
clinically relevant pathogens were correctly detected.
Discussion
The single-tube LATE-PCR multiplex assay described
above can detect 17 microbial and fungal pathogens using
genomic DNA. These 17 pathogens were chosen based on
discussions with our clinical collaborators at the University of California at Davis and took the clinical prevalence
of pathogens and mixtures associated with septicaemia
into account. Each of the 17 targets was detected by its
specific pattern of hybridization to sequence-specific low
temperature probes. This multiplex assay was also shown
to resolve complex mixtures constructed with genomic
DNA.
Currently, there are several septicaemia assays that have
been described and evaluated for clinical relevance (Andrade et al. 2008; Lehmann et al. 2008; Louie et al. 2008;
Dierkes et al. 2009; Westh et al. 2009; Zhao et al. 2009;
Chakravorty et al. 2010; Bravo et al. 2011). The three
most pertinent assays are the SeptiFast assay (Lehmann
et al. 2008), a real time iso-thermal assay (Zhao et al.
2009) and a LATE-PCR Molecular Beacon Assay (Chakravorty et al. 2010). All three of these assays provide
useful clinical information for bacterial and fungal pathogen detection, but all of these tests use multiple tubes
and none is highly multiplexed into a single-tube assay as
described here. Moreover, as in the case of many other
assays for sepsis that have been described, all three of
these assays have limitations in terms of ease of use and
clinical breath.
The SeptiFast (Lehmann et al. 2008) assay uses three
separate symmetric PCR reaction tubes to differentiate
gram-positive bacteria, gram-negative bacteria and fungal
pathogens using the internal transcribed spacer region
between the 16S and 23S ribosomal DNA sequences for
bacteria and the 18S and 5.8S ribosomal sequences for
fungi. The assay is prone to the detection of reaction
mixture contamination components from unwanted bacteria and the assay uses a cutoff point in cycle threshold
(Ct) value to limit detection of low background contamination. This limitation leads to low sensitivity vs blood
culture analysis of certain pathogen targets (Lehmann
et al. 2008).
The isothermal assay (Zhao et al. 2009) uses the 16S and
28S ribosomal DNA in two reaction tubes for bacterial and
fungal detection, and is prone to the same risk of contamination and sensitivity levels as the SeptiFast assay. The
Journal of Applied Microbiology 114, 457--469 © 2012 The Society for Applied Microbiology
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L.M. Rice et al.
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Figure 2 The use of two dye channels for the detection of 1–99% mixtures. Solid black lines 100% Staphylococcus aureus for all panels.
(a) CalOrg560 and FAM fluorescent contours show the Staph. aureus at the 1% level (grey a1) in a background of 99% Klebsiella pneumoniae
(grey a2); the reverse mixture is shown by Kl. pneumoniae at the 1% level (black dashed a2) in a background of 99% Staph. aureus (black
dashed a1). (b) CalOrg560 and CalRed610 fluorescent contours of Staph. aureus at the 1% level (grey b1) in a background of 99% Pseudomonas aeruginosa (grey b2); the reverse mixture is shown by Ps. aeruginosa at the 1% level (black dashed b2) in a background of 99% Staph. aureus
(black dashed b1). (c) The CalOrg560 and Quasar670 fluorescent contour shows the detection of the mixture of Staph. aureus at the 1% level
(grey c1) in a background of 99% Enterococcus faecalis (grey c2); the reverse mixture shows detection of Enterococcus faecalis at the 1% level
(black dashed c2) in a background of 99% Staph. aureus (black dashed c1).
LATE-PCR molecular beacon assay (Chakravorty et al.
2010) also targets the 16S ribosomal DNA region for bacterial targets and uses six tubes to accomplish the discrimination of over 100 bacterial signatures, where each tube is
designated to resolve a single fluorescent probe. This assay
has similar issues as other 16S assays with regard to reaction mixture contamination detection and sensitivity. The
need to split a blood sample of low pathogen concentration
into six tubes will cause sensitivity issues.
The LATE-PCR single-tube multiplex assay described
here expands on the three assays described above and
also shows some advantages and limitations. Unlike the
16S based assays, this assay only achieves designed results.
Consequently, the assay will not pick up unknown patho-
gens outside of the genera or species that the assay was
designed to detect. This result is actually an advantage in
that low levels of contamination in reaction mixture
components are not picked up. It is also a limitation in
that adding more pathogens to be detected in these four
dye channels is not prudent. However, expanding
the number of dye channels would be a possibility for
the inclusion of more pathogen detection. As shown in
the results section, the assay detects mixtures of pathogens cleanly, whether they occur in a single dye channel
or in multiple dye channels.
The greatest advantage of the multiplex assay described
here is that all bacterial and fungal pathogens are
detected in a single tube using only four fluorescent dye
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Figure 3 Shows the detection of complex mixture in a single dye channel. Fluorescent contours (a1, b1) were normalized as described in Materials and methods. The corresponding fluorescent signatures (a2, b2) show that the inflection points of the mixtures are the same as the positive
controls. (a1) In CalOrg560 the detection of the positive controls: Staphylococcus aureus (solid black line) and Enterobacter aerogenes (solid grey
line) and the mixture of 1% Staph. aureus, in a background of 99% Ent. aerogenes (dotted black line). (b1) In CalRed610 the detection of the
positive controls Pseudomonas aeruginosa, (solid black line) and of Staphylococcus hominis (coagulase-negative staphylococcus – solid grey line)
and the mixture of 1% Ps. aeruginosa, in a background of 99% Staphylococcus hominis (dotted line).
600
400
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60
50
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Figure 4 Full Multiplex mixtures of three genomic DNA targets, Pseudomonas aeruginosa, Staphylococcus hominis, in CalRed610 (a1) and Candida glabrata in Quasar 670 (a2). The fluorescent signatures are shown. The solid lines are positive controls. The black line represents an equal mixture at 1000 copies each, while the solid grey line represents an equal mixture of 100 000 copies. (a1) In the CalRed610 channel the dashed
black line illustrates a mixture of 1% Ps. aeruginosa in a background of 99% Staph. hominis and C. glabrata, the dashed grey line – a mixture
of 1% Staph. hominis in a background of 99% Ps. aeruginosa and C. glabrata. (a2) In the Quasar670 channel, the dashed black line – 1%
C. glabrata in a background of 99% Staph. hominis and Ps. aeruginosa.
channels. This saves dividing precious amounts of targets
over several tubes for the analysis. We have also shown
that we can easily analyse related species under a single
genus for example Candida spp. (P450). The assay still
could be further optimized to lower the limit of detection
values that are shown in Table 4. Further clinical samples
468
also need to be tested to determine the breath of detection of this assay. The accompanying article discusses a
preliminary evaluation of this LATE-PCR single-tube
multiplex assay. We conclude that the multiplex assay
described here holds promise as a rapid and accurate
molecular diagnostic screening method for septicaemia.
Journal of Applied Microbiology 114, 457--469 © 2012 The Society for Applied Microbiology
L.M. Rice et al.
Acknowledgements
We are grateful to Dr Barry Kreiswirth of PHRI, Newark N.
J. for genomic DNA from bacterial cultures of 12 strains of
Staph. aureus. We also thank the UC Davis POC Technologies Center for providing ATCC genomic DNA samples and
Anna Dillier and Samaan Mahmoudzadeh of the Kost Lab
for their thorough input and review of this article. This
research was supported by the NIH grants (1RC1EB01054301 and 5RC1EB010643-02) and also by Smiths Detection
Diagnostics, Inc., Watford, UK.
References
Andrade, S.S., Bispo, P.J.M. and Gales, A.C. (2008) Advances
in the microbiological diagnosis of sepsis. Shock 30, 41–46.
Balk, R.A., Ely, E.W. and Goyette, R.E. (2001) Sepsis
Handbook. Vanderbilt University Medical Center: National
Initiative in Sepsis Education.
Bravo, D., Blanquer, J., Tormo, M., Aquilar, G., Borras, R.,
Solano, C., Clari, M.A., Costa, E. et al. (2011) Diagnostic
accuracy and potential clinical value of the LightCycler
SeptiFast assay in the management of bloodstream
infections occurring in neutropenic and critically ill
patients. Int J Infect Dis 15, 326–331.
Chakravorty, S., Aladegbami, B., Burday, M., Levi, M., Marras,
S.A.E., Shah, D., El-Hajj, H.H., Kramer, F.R. et al. (2010)
Rapid universal identification of bacterial pathogens from
clinical cultures by using a novel sloppy molecular beacon
melting temperature signature technique. J Clin Microbiol
48, 258–267.
Dhanasekaran, S., Doherty, T. M., Kenneth, J. and TB Trials
Study Group (2010) Comparison of different standards for
real-time PCR-based absolute quantification. J Immunol
Methods 354, 34–39.
Dierkes, C., Ehrenstein, B., Siebig, S., Linde, H.J., Reischl, U.
and Salzberger, B. (2009) Clinical impact of a
commercially available multiplex PCR system for rapid
detection of pathogens in patients with presumed sepsis.
BMC Infect Dis 9, 125.
Gentile, N.L., Dillier, A.M., Williams, G.V., Ackers, J., Reis, A.
H. Jr, Rice, L.M., Wangh, L.J., Czajka, J. and Kost, G.J.
(2012) Verification of monoplex and multiplex LATE-PCR
gene-specific sepsis assays using clinical isolates. J Appl
Microbiol, in press. doi: 10.1111/jam.12062.
Hartshorn, C., Anshelevich, A. and Wangh, L.J. (2005a) Laser
zona-drilling does not induce hsp70i transcription in blastomeres of 8-cell mouse embryos. Fertil Steril 84, 1547–1550.
Hartshorn, C., Anshelevich, A. and Wangh, L.J. (2005b)
Rapid, single-tube method for quantitative preparation
and analysis of RNA and DNA in samples as small as one
cell. BMC Biotechnol 5, 2.
Hartshorn, C., Eckert, J.J., Hartung, O. and Wangh, L.J.
(2007) Single-cell duplex RT-LATE-PCR reveals Oct4 and
LATE-PCR multiplex assay for sepsis
Xist RNA gradients in 8-cell embryos. BMC Biotechnol 7,
87.
Lehmann, E.L., Hunfeld, K.P., Emrich, T., Haberhausen, G.,
Wissing, H., Hoeft, A. and St€
uber, F. (2008) A multiplex
real-time PCR assay for rapid detection and differentiation
of 25 bacterial and fungal pathogens from whole blood
samples. Med Microbiol Immunol 197, 313–324.
Louie, R.F., Tang, Z., Albertson, T.E., Cohen, S.S. and Tran,
N.K. (2008) Multiplex polymerase chain reaction detection
enhancement of bacteremia and fungemia. Crit Care Med
36, 1487–1492.
Osborne, A., Reis, A.H. Jr, Bach, L. and Wangh, L.J. (2009) Singlemolecule LATE-PCR analysis of human mitochondrial
genomic sequence variations. PLoS One 4, e5636.
Pierce, K.E., Sanchez, J.A., Rice, J.E. and Wangh, L.J. (2005)
Linear-After-The-Exponential (LATE)-PCR: primer design
criteria for high yields of specific single-stranded DNA
and improved real-time detection. Proc Natl Acad Sci USA
102, 8609–8614.
Pierce, K.E., Mistry, R., Reid, S.M., Bharya, S., Dukes, J.P. and
Wangh, L.J. (2009) Design and optimization of a novel
reverse transcription linear-after-the-exponential PCR for
the detection of foot-and-mouth disease virus. J Appl
Microbiol 109, 180–189.
Rice, J.E., Sanchez, J.A., Pierce, K.E., Reis, A.H. Jr, Osborne, A.
and Wangh, L.J. (2007) Monoplex ⁄ multiplex linearafterthe-exponential-PCR assays combined with PrimeSafe
and Dilute-’N’-Go sequencing. Nat Protoc 2, 2429–2438.
Ronish, B., Hakhverdvan, M., St
ahl, K., Gallardo, C.,
Fernandez-Pinero, J., Belak, S., Leblanc, N. and Wangh, L.
J. (2010) Design and verification of a highly reliable
Linear-After-The-Exponential PCR (LATE-PCR) assay for
the detection of African swine fever virus. J Virol Methods
172, 8–15.
Salk, J.J., Sanchez, J.A., Pierce, K.E., Rice, J.E., Soares, K.C.
and Wangh, L.J. (2006) Direct amplification of singlestranded DNA for pyrosequencing using linear-after-theexponential (LATE)-PCR. Anal Biochem 353, 124–132.
Sanchez, J.A., Pierce, K.E., Rice, J.E. and Wangh, L.J. (2004)
Linear-After-The-Exponential (LATE)-PCR: an advanced
method of asymmetric PCR and its uses in quantitative
real-time analysis. Proc Natl Acad Sci USA 101, 1933–1938.
Sanchez, J.A., Abramowitz, J.D., Salk, J.J., Reis, A.H. Jr, Pierce,
K.E., Rice, J.E. and Wangh, L.J. (2006) Two-temperature
LATE-PCR endpoint genotyping. BMC Biotechnol 6, 44.
Westh, H., Lisby, G., Breysse, F., B€
oddinghaus, B., Chomarat,
M., Gant, V., Goglio, A., Raglio, A. et al. (2009) Multiplex
real-time PCR and blood culture for identification of
bloodstream pathogens in patients with suspected sepsis.
Clin Microbiol Infect Dis 15, 544–551.
Zhao, Y., Park, S., Kreiswirth, B.N., Ginocchio, C.C., Veyret,
R., Laayoun, A., Troesch, A. and Perlin, D.S. (2009) Rapid
real-time nucleic acid sequence-based amplificationmolecular beacon platform to detect fungal and bacterial
bloodstream infections. J Clin Microbiol 47, 2067–2078.
Journal of Applied Microbiology 114, 457--469 © 2012 The Society for Applied Microbiology
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