Clinica Chimica Acta 363 (2006) 206 – 220

www.elsevier.com/locate/clinchim

Review

Nucleic acid-based methods for the detection of bacterial pathogens:

Present and future considerations for the clinical laboratory
Elizabeth A. Mothershed *, Anne M. Whitney
Meningitis and Special Pathogens Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases,
Centers for Disease Control and Prevention, Atlanta, GA 30333, United States

Received 3 April 2005; received in revised form 25 May 2005; accepted 26 May 2005
Available online 1 September 2005

Abstract

Background: Recent advances in nucleic acid-based methods to detect bacteria offer increased sensitivity and specificity over traditional
microbiological techniques. The potential benefit of nucleic acid-based testing to the clinical laboratory is reduced time to diagnosis, high
throughput, and accurate and reliable results.
Methods: Several PCR and hybridization tests are commercially available for specific organism detection. Furthermore, hundreds of nucleic
acid-based bacterial detection tests have been published in the literature and could be adapted for use in the clinical setting. Contamination
potential, lack of standardization or validation for some assays, complex interpretation of results, and increased cost are possible limitations
of these tests, however, and must be carefully considered before implementing them in the clinical laboratory.
Conclusions: A major area of advancement in nucleic acid-based assay development has been for specific and broad-range detection of
bacterial pathogens.
Published by Elsevier B.V.

Keywords: Molecular diagnostics; Clinical laboratory; PCR; Real-time PCR; Bacterial pathogens; NATs

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
2. Detection of a specific bacterial pathogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
2.1. Cycling amplification technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
2.1.1. PCR, real-time PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
2.1.2. Nested PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
2.1.3. PCR-ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
2.1.4. Ligase chain reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
2.2. Isothermal and other amplification technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
2.2.1. Nucleic acid sequence-based amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
2.2.2. Transcription-mediated amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
2.2.3. Strand displacement amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
2.2.4. Rolling circle amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
2.2.5. Cycling probe technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
2.2.6. Branch DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
2.2.7. Hybrid capture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

* Corresponding author.
E-mail address: ekm9@cdc.gov (E.A. Mothershed).

0009-8981/$ - see front matter. Published by Elsevier B.V.

doi:10.1016/j.cccn.2005.05.050
 E.A. Mothershed, A.M. Whitney / Clinica Chimica Acta 363 (2006) 206 – 220 207

3. Detection of bacterial pathogens by multiple targets or universal targets. . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

3.1. Multiplex PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
3.2. Microarray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
3.3. Sequencing-based identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
4. Detection of bacterial pathogens by nucleic acid hybridization or mass spectrometry . . . . . . . . . . . . . . . . . . . . . 213
4.1. Fluorescence in situ hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
4.2. Peptide nucleic acid-FISH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
4.3. Line probe assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
4.4. Hybridization protection assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
4.5. Mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
5. Quality control: nucleic acid extraction and contamination prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
6. How to choose which test is right for your laboratory? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
7. Future trends for NATs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217

1. Introduction in culture-negative specimens. NATs for specific organ-

Successful treatment of a patient with a bacterial
infectious disease requires rapid and specific identification
of the causative agent. Identification of bacterial pathogens
by traditional methods is still a crucial element of the
diagnostic process. However, methods such as culturing
and sub-culturing organisms, especially those that are
fastidious, culturing clinical specimens taken after anti-
biotic treatment, determining antimicrobial susceptibility of
the organism, and biochemical testing can be laborious and
time-consuming, and may prolong definitive diagnoses and
treatment of the patient. While there has been improvement
in traditional methods, such as automation of blood culture
systems [1], clinical laboratories have begun to adopt
nucleic acid-based tests (NATs) to identify pathogens
rapidly and reliably.
The first NAT cleared for use by the Food and Drug
Administration (FDA) was the Gen-Probe PACE test
(1988) that used nucleic acid hybridization to detect
Chlamydia and gonococci (http://www.gen-probe.com/
prod_serv/std_pace.asp). A technological breakthrough in
molecular biology came in 1983 with the development of
polymerase chain reaction (PCR) [2]. In subsequent years,
nucleic acid amplification tests (NAATs), including PCR-
based assays, were developed to detect virtually every
clinically relevant bacterial pathogen. (Note: going forward
the term NATs will be used to describe all nucleic acid
based tests, including NAATs.)
The advantages of NATs over microbiologic methods
include rapid results, low detection limits (theoretically a
single cell), and specific organism detection. In a hospital
setting, rapid pathogen detection is important for faster
and improved patient treatment and potentially shorter
hospital stays. Community outbreaks and nosocomial
infections may be thwarted if early pathogen identification
results in patient isolation and/or directed prophylaxis of
patient contacts. NATs can be used to detect the presence
of organisms directly in clinical specimens without
culture, in cultures with abbreviated incubation times, or
isms such as Mycobacterium tuberculosis, Chlamydia
trachomatis, and Neisseria gonorrhoeae [3] are available
from several manufacturers, some of which are listed in
Table 1. In addition, hospital infection control and
epidemiology programs are benefiting from the use of
NATs for detecting antibiotic resistance genes and for sub-
typing bacteria.
While NATs offer many advantages for the clinical
laboratory, care must be exercised when using these tests.
Because of the increased sensitivity of NATs, contami-
nation prevention and quality control must be imple-
mented. Theoretically, in a NAAT, one copy of a target
gene will be amplified; therefore, if the one copy is from
a laboratory contaminant or previous experiment, a false
positive result will be observed. Conversely, inhibitors in
clinical specimens or DNA degradation can lead to false
negative results. Another disadvantage is that NATs
detect nucleic acids but do not indicate viability of the
pathogen.
In this review, we will discuss the identification or
characterization of bacterial pathogens using NATs for a
single nucleic acid target, multiple targets, or universal
targets. Furthermore, we will review NATs that use hybrid-
ization technologies or mass spectrometry. Quality control
and contamination prevention strategies, as well as criteria
to consider when choosing a NAT will be discussed. Finally,
future trends in NATs technology and methods will be
presented.
2. Detection of a specific bacterial pathogen
2.1. Cycling amplification technologies
2.1.1. PCR, real-time PCR and RT-PCR
Amplification of a target sequence by conventional
PCR using two primers is typically detected and
visualized by gel electrophoresis using DNA-binding
fluorescent dyes. By comparison, real-time PCR uses a
 208
Table 1
Commercially available diagnostic tests for detection of common bacterial pathogens
Organism detected Test name Method/technology Time requirement Sensitivity Specificity Sample type FDA Manufacturer References

E.A. Mothershed, A.M. Whitney / Clinica Chimica Acta 363 (2006) 206 – 220
for testing (%) (%) clearance
Chlamydia trachomatis HC2 CT ID DNA/RNA Hybrid 4h 96 100 Endocervical swabs Yes Digene [26]
Capture
PACE2 CT HPA NA 78 99 Endocervical swabs Yes Gen-Probe [100]
APTIMA CT TMA/16S target NA 96 – 97 97 – 99 Urine or urethral swab No Gen-Probe [9]
Neisseria gonnorhoeae COBAS AMPLICOR PCR 4–8 h 92.4 99.5 Endocervical swabs Yes Roche [101]
BDProbeTec ETa SDA 1–2 h 99.2 99.3 Urine Yes Becton Dickinson [102]
NucliSens Basic Kitb NASBA 8h 97.9 98.7 Urethral or cervical swab No bioMerieux [12]
Mycobacterium tuberculosis Amplified MTD Direct test TMA 3.5 h 91c 98 Sputum Yes Gen-Probe [103]
BD Probe tec ET SDA 1–2 h 93 – 95 92 – 100 Respiratory or No Beckton – Dickinson [17 – 19,22,104]
non-respiratory
COBAS AMPLICORa PCR 4–8 h 97 100 Respiratory or No Roche [8]
non-respiratory
MRSA IDI-MRSA Real-time PCR 1.5 h 91.7 93.5 Nasal swabs Yes Infectio Diagnostics, Inc. [105]
Velogene-CPT Rapid MRSAd CPT 90 min 98 100 Culture Yes ID Biomedical, Inc. [24]
Group A Streptococci GAS Direct Test HPA 24 h 94.8 100 Pharyngeal swab No Gen-Probe [106]
Group B Streptococci IDI-Strep B Real-time PCR 1.5 h 94 95.9 Vaginal swabs Yes Infectio Diagnostics, Inc. [107]
Gardnerella, Trichomonas BD Affirm VPIII Microbial Hybridization 45 min 82 – 95e 98 – 100e Vaginal swabs Yes Becton Dickinson, Inc. [108,109]
vaginalis, and Candida spp. Identification Test
Escherchia coli O157:H7 BAX System PCR 8h 99 99 Water No Qualicon, Inc. [110]
HPA, hybridization probe assay; TMA, transcription mediated amplification; SDA, strand displacement amplification; NASBA, nucleic acid sequence based amplification, CPT, cycling probe technology; NA, not
available.
a
Not available in the US.
b
Primers designed by individual laboratory.
c
Sensitivity improved from 61% to 91% after retest and implementation of protocol for inhibition detection.
d
No longer available.
e
From manufacturer’s website (www.bd.com).
 E.A. Mothershed, A.M. Whitney / Clinica Chimica Acta 363 (2006) 206 – 220
fluorescently labeled probe with two flanking primers in
the reaction. Real-time PCR offers many advantages over
conventional PCR such as increased sensitivity and
rapidity, broader dynamic range, elimination of post-
amplification handling steps, and higher throughput
conducive to automation [4]. Real-time PCR assays may
use the intercalating fluorescent dye, SYBR\ Green, dual-
labeled probes (TaqMan\), hybridization probes (Light-
Cycler), Molecular Beacons, or Scorpionsi as the means
of detecting amplification. Some recent publications using
real-time PCR for bacterial detection are listed in Table 2.
Use of a closed system greatly minimizes carry-over
contamination and most real-time PCR formats offer the
option of melting curve analysis, which allows the
amplification product to be discriminated from nonspecific
product or primer – dimers.
Most NATs for bacterial detection are DNA-based
because the genomes of bacteria are DNA, rather than
RNA as with some viruses. However, reverse-transcriptase
PCR (RT-PCR)-based tests for bacteria have been published
such as the one for the detection of hemolysin from Vibrio
parahaemolyticus described by Nakaguchi et al. [5]. RT-
PCR is a method for studying gene expression, and uses
RNA as its template to produce complementary DNA
(cDNA). The cDNA is then amplified by PCR. RT-PCR and
real-time PCR technologies are commonly used by research
laboratories and some clinical laboratories, and many
commercially available NATs for specific pathogen detec-
tion utilize these methods (Table 1). Sensitivities and
specificities of these tests, however, may be affected by
the specimen type used, nucleic acid extraction method, and
quality of the primers and fluorescent probes used in the
tests.
Though real-time PCR is in widespread use because of
its enhanced sensitivity, the overriding limitation of this
technology is the high cost of special reagents and
instrumentation. The price of a single real-time PCR
reaction including DNA extraction can cost up to $10,
while conventional PCR costs less than $3 per reaction.
Instrumentation costs range from $24,000 for the Strata-
gene MX3000 (La Jolla, CA), for example, to over
$130,000 for an automated, high-throughput instrument
Table 2
Recently published real-time PCR assays for detecting bacterial pathogens
Organism Platform Detection
Leptospira spp. LightCycler\
SYBR Green I
Neisseria meningitidis TaqMan\ Dual-labeled probe
Burkholderia TaqMan\ Dual-labeled probe
pseumomallei
Bordatella pertussis LightCycler\ Hybridization probes
Bordetella parapertussis LightCycler\ Hybridization probes
Escherichia coli LightCycler\ Hybridization probes
Methicillin-resistant SmartCycler\ Molecular Beacons
Staphylococcus aureus
Group B Streptococcus SmartCycler\ Molecular Beacons
Clostridium difficile SmartCycler\ Molecular Beacons
209
like the ABI 7900HT (Foster City, CA). Less expensive
instruments such as the Roche LightCycler\ and Cepheid
SmartCycler\ could be options for some laboratories,
however these platforms are limited by the number of
reactions (16 – 32) that can be tested per run. Real-time
PCR primer and probe design may be confounding since a
small amplicon (< 500 base pairs) is preferable. Post-
amplification manipulation of the amplicon, such as
cloning or sequencing, may also be difficult. Finally, the
inability to determine amplicon size could be viewed as a
disadvantage by some investigators [6].
2.1.2. Nested PCR
Nested PCR is a conventional PCR method that amplifies
a target region of DNA with an outer primer pair in an initial
reaction, followed by a second amplification using an
internal primer pair. It is useful for pathogen detection in
clinical specimens because of its enhanced sensitivity over a
single amplification, but can be problematic due to carry-
over contamination from the first reaction to the second [7].
Nested PCR has also been used for the detection of the 16S
and 23S rRNA genes from a variety of bacteria and provides
multiple overlapping amplicons for accurate sequencing of
these genes (see Section 3.3).
2.1.3. PCR-ELISA
The PCR – enzyme linked immunosorbent assay (ELISA)
format is a viable alternative to real-time PCR methods. PCR
products are labeled (e.g., by digoxigenin) during amplifi-
cation and a capture probe specific to the PCR amplicon is
used to immobilize the amplicon to a well of a microtiter
plate. An enzyme-linked antibody targeting the label (e.g.,
anti-digoxigenin) is then used to quantitate PCR products.
As Yam et al. demonstrate, a biotinylated PCR-ELISA for
direct detection of M. tuberculosis using a single-tube nested
PCR method provides a simple, accurate, high throughput
test with sensitivity and specificity comparable to the
commercial, PCR-based COBAS AMPLICOR system
(Roche Diagnostics; Table 1) at around one-fourth the cost
[8].
2.1.4. Ligase chain reaction
Ligase chain reaction (LCR) is a DNA amplification
technique, but differs from PCR because it does not produce
amplicon through polymerization of nucleotides. In LCR, a
Reference primer is synthesized in 2 fragments and annealed to the
[111] template. The ligase enzyme will join the 2 fragments only if
[112] they match exactly to the template sequence. Subsequent
[113] PCR reactions will amplify the template only if the primer
fragments are joined. A once-popular commercial test, the
[114]
LCx (Abbott Laboratories, Abbott Park, IL) for Chlamydia
[114]
[115] detection, was apparently taken off the market in 2003 due to
[105] problems with negative controls (http://www.chlamydiae.
com/restricted/docs/labtests/diag_lcr.asp), but its perform-
[107] ance has been compared to other commercial tests in the
[116]
literature [9].
210 E.A. Mothershed, A.M. Whitney / Clinica Chimica Acta 363 (2006) 206 – 220
2.2. Isothermal and other amplification technologies
2.2.1. Nucleic acid sequence-based amplification
Nucleic acid sequence-based amplification (NASBA) is
an isothermal, transcription-based amplification method
which amplifies RNA from either an RNA or DNA target
and utilizes avian myeloblastosis virus reverse transcrip-
tase, RNase H and T7 RNA polymerase. NASBA has been
used to detect various bacterial pathogens including
Escherichia coli and Mycoplasma pneumoniae [10,11].
NASBA has demonstrated equivalent or improved sensi-
tivity to PCR-based methods and has the potential
advantage of being easier to optimize than conventional
PCR [12]. Rodriguez-Lazaro et al. describe a NASBA
assay for detecting the dnaA gene of Mycobacterium
avium subsp. paratuberculosis from water and milk and
detail the inclusion of an internal amplification control
specific for the NASBA method [13,14]. Diagnostic tests
using NASBA are also commercially available for the
pathogens C. trachomatis and N. gonorrhoeae (Table 1).
BioMerieux (Durham, NC) has combined NASBA and
Molecular Beacons into a test system called EasyQ for
simultaneous amplification and fluorescent detection of
specific organisms (http://www.biomerieux-usa.com/clinical/
nucleicacid/easyq/easyq_technology.htm).
2.2.2. Transcription-mediated amplification
Transcription-mediated amplification (TMA) is another
isothermal amplification method that can be used to target
either DNA or RNA. TMA uses RNA transcription (RNA
polymerase) and DNA synthesis (reverse transcriptase) to
produce an RNA amplicon from a target nucleic acid. Since
RNA is more labile than DNA in the laboratory environ-
ment, this feature diminishes the possibility of carry-over
contamination. TMA produces 100– 1000 copies per cycle
as compared to PCR and LCR that produce only 2 copies
per cycle. This results in a 10 billion-fold increase of copies
within about 15 – 30 min. TMA has gained popularity in the
clinical laboratory with the development of commercial tests
including the APTIMA tests for the detection of C.
trachomatis and N. gonnorhoeae (Table 1) [15].
In a comparison of Gen-Probe APTIMA CT and GC
assays with the APTIMA combo 2 assay, the Abbott LCx
assay, direct fluorescent antibody test, and culture for the
detection of C. trachomatis and N. gonorrhoeae, Boy-
adzhyan et al. reported that the APTIMA tests detected
more confirmed positive specimens than culture, direct
fluorescent antibody test, or LCx [9]. A similar study found
the Abbott LCx, BD ProbeTec ET and the Gen-Probe
APTIMA Combo 2 to be very sensitive (96%, 96% and
100%, respectively) and specific (99%, 100% and 99%,
respectively) for detection of C. trachomatis in urine [16].
Another Gen-Probe product that incorporates TMA is the
amplified M. tuberculosis direct test (MTD). Lemaitre et al.
compared real-time PCR to the AMTDII test (Gen-Probe,
San Diego, CA) and found that real-time PCR was only
slightly less sensitive to inhibitors than the AMTDII [17].
Gamboa et al. found that, compared to culture and staining,
the AMTDII test was 95% and 100% sensitive and specific,
respectively, using respiratory specimens, and slightly less
sensitive using non-respiratory specimens [18]. Kerleguer et
al. found the MTD test to be 93% sensitive and 100%
specific using lymph node aspirates, while culture was 89%
sensitive [19].
2.2.3. Strand displacement amplification
Strand displacement amplification (SDA), first
described in 1992 [20], is an isothermic amplification
method in which a primer containing a restriction site is
annealed to the DNA template. Next, amplification
primers are annealed to 5V adjacent sequences (forming a
nick) to begin amplification. Newly synthesized DNA is
nicked by the corresponding restriction enzyme and the
polymerase starts amplification again, displacing the newly
synthesized strands. In a single reaction, 109 copies of
target DNA can be produced. SDA is the basis for some
commercial detection tests such as BDProbeTec (Becton
Dickinson, Franklin Lakes, NJ) and has been evaluated
recently for the identification of M. tuberculosis directly
from clinical specimens [21]. A study of BDProbeTec’s
assay for C. trachomatis and N. gonorrhoeae on vaginal
swab specimens showed sensitivity and specificity equiv-
alent to those of PCR for the detection of C. trachomatis
and superior to those of culture for the detection of N.
gonorrhoeae [22].
2.2.4. Rolling circle amplification
In rolling circle amplification (RCA), a single forward
primer is extended by DNA polymerase along a circular
template for many rounds, displacing upstream sequences
and producing a long single-stranded DNA of multiple
repeats. The linear RCA reaction can run for several hours
or days, producing millions of copies of the small circle
sequence. In exponential RCA, a primer pair is used. The
second primer targets the single-stranded DNA product of
the first primer and initiates hyper-branching in the DNA
replication, creating as many as 1012 copies/h. As is the
case for all isothermal technologies, there is no need for
special instrumentation, since temperature cycling is not
required. A major advantage of RCA is that, unlike PCR,
this technology is resistant to contamination and, unlike
some other isothermal technologies, requires little or no
assay optimization. A recent review discusses details of the
technology and the latest progress in RCA diagnostics
[23].
2.2.5. Cycling probe technology
Cycling probe technology (CPT) is an isothermal probe
amplification system for detection of target DNA that
utilizes a RNA – DNA chimeric probe (RNA sequence
flanked by two DNA sequences) to hybridize to a specific
region of an amplified gene. Once hybridized, the internal
 E.A. Mothershed, A.M. Whitney / Clinica Chimica Acta 363 (2006) 206 – 220
RNA part of the probe is cleaved by RNase H, which
recognizes specifically the RNA –DNA duplex. Once the
probe is cleaved, the reporter dye and quencher dye on each
side of the probe are separated and fluorescence is emitted.
The fluorescence signal increases proportionally as the
probe is cleaved, allowing for measurement of the amplified
product. The probe-based Velogene Rapid MRSA Identi-
fication Assay (ID Biomedical Corp., Vancouver, British
Columbia, Canada) has been used previously for detection
of methicillin-resistant Staphylococcus aureus (MRSA), but
is no longer available in the US [24].
2.2.6. Branched DNA
Another technique that utilizes signal rather than target
amplification is called branched DNA (bDNA). In bDNA,
many branched, labeled DNA probes generate signal via
alkaline phosphatase upon binding to a specific nucleic acid
target. bDNA has been used to detect the mecA gene in
MRSA [25], though it is more commonly used for
determination of viral load.
2.2.7. Hybrid capture
Signal amplification is also the basis for some
commercial tests such as the Hybrid Capture (HC2)
assays from Digene (Gaithersburg, MD) (Table 1). In the
Hybrid Capture tests, a lysis solution is used to release
DNA from the bacteria, and specific RNA probes combine
with the target DNA to form a hybrid. The RNA/DNA
hybrid is then captured by specific antibodies to a solid
support, and the hybrids are detected by a second antibody
conjugated to alkaline phosphatase which cleaves a
chemiluminescent substrate to release light. Because many
antibody molecules attach to each hybrid, the signal is
amplified. Hybrid Capture systems are available to detect
C. trachomatis, N. gonorrhoeae, human papillomavirus,
cytomegalovirus and hepatitis B. A benefit to using a
signal amplification assay versus a target amplification
assay (e.g. PCR) is that amplicons are not produced in the
laboratory; therefore, the chance of cross-contamination of
subsequent reactions is reduced. Apparently, sensitivity is
not lost with signal amplification assays. Independent
studies have found that the Digene Hybrid Capture II kit
was a highly sensitive and specific assay for detection of
C. trachomatis [26, 27].
3. Detection of bacterial pathogens by multiple targets or
universal targets
The overriding disadvantage of organism-specific assays
is that they are limited in scope and useful only when a
particular agent is suspected. For this reason, technologies
such as multiplex PCR, microarray, and broad-range PCR
assays have been developed for the purpose of testing
simultaneously for more than one organism or as a means to
screen clinical specimens for bacterial etiologic agents.
211
3.1. Multiplex PCR
Multiplex PCR utilizes more than one set of primers in a
reaction and can be used for the simultaneous detection of
multiple bacterial pathogens. Either conventional or real-
time PCR can be used for a multiplex reaction. The Hyplex
Blood Screen multiplex PCR-ELISA system (BAG, Lich,
Germany) is a commercially available test used to screen
positive blood cultures for the most common bacterial
pathogens that cause sepsis. This test system uses PCR to
amplify organism-specific housekeeping genes and detects
the amplification products by hybridization to oligonucleo-
tide probes in an ELISA format [28]. Also, multiplex PCR
in combination with colorimetric Covalink NH microwell
plate (DNA – DNA sandwich hybridization) detection is a
method used in clinical settings because of its low cost and
ease of use. As Lee et al. show by simultaneously detecting
Vibrio spp. and Salmonella enterica in shellfish, traditional
multiplex PCR with multiple primer sets can also demon-
strate a high level of sensitivity and specificity [29].
Multiplex PCR is also useful for detecting a species-specific
target and a serogroup-specific target in a single reaction.
For example, Taha has developed a multiplex PCR for the
detection of Neisseria meningitidis in which 6 pairs of
serogroup-specific primers are added together in a single
reaction [30].
The Luminex system (Luminex Corp., Austin, TX)
uses a recently developed bead-based technology, xMAP,
which has the potential for routine testing in the clinical
laboratory. Introduced as an immunoassay, this technology
has gained popularity in a nucleic acid-based format
because of the capability to detect many targets in a
single test (multiplex), while still maintaining a high
level of sensitivity and specificity [31]. Polystyrene
microbeads are imbued with two fluorescent dyes. The
ratio of one dye to another creates 100 unique spectral
signatures. These bead sets can be coated with peptides,
receptors, oligonucleotides or antibodies. In a recent
study, Dunbar et al. demonstrated simultaneous detection
of multiple food-borne pathogens, E. coli, Salmonella,
Listeria monocytogenes and Campylobacter jejuni with
rapid (40 min), specific identification; however, sensitivity
was not ideal, because the test required 106 to 107
genome copies for detection [32]. As with any method,
though, multiplex PCR is not without disadvantages.
Problems such as low sensitivity [33], cross-reactivity,
and preferential binding of SYBR\ Green I to longer,
higher G + C% amplification products [34] have been
reported for multiplex reactions.
3.2. Microarray
Microarray refers to a small, two-dimensional high-
density matrix of DNA fragments which are printed or
synthesized on a glass or silicon slide (chip) in a specific
order. Hybridization of the DNA fragments to fluorescently
212 E.A. Mothershed, A.M. Whitney / Clinica Chimica Acta 363 (2006) 206 – 220

labeled probes is detected by advanced instrumentation and Table 3

software. However, microarrays for bacterial diagnostics Bacterial pathogens identified or characterized by 16S rRNA gene
sequencing
have not been incorporated into routine testing in clinical
Organism Syndrome or Specimen/site Reference
laboratories because of the complex technical aspects for
disease of isolation
successful experiments such as uniform stringency over the
Atopobium vaginae Bacterial vaginosis Vaginal lavage [117]
array chip, data analysis and interpretation, and high cost.
Bacillus anthracis Respiratory and Various sitesa [45]
Even with such confounders, some manufacturers offer ‘‘off cutaneous anthrax
the shelf’’ assays which are reported to be sensitive, specific, Bacillus cereus Pneumonia Sputum, blood [118]
and easy-to-interpret. Affymetrix has developed the Brucella spp. Brucellosis Various sitesb [49]
Advanced Array Technology (AAT, Eppendorf Array Tech- Burkholderia Melioidosis Various sitesc [50]
pseudomallei
nologies, Belgium) Staphychip which detects 15 species of
Clostridium hathewayi d Septic shock Blood [119]
Staphylococcus, including methicillin-resistant S. aureus Corynebacterium Sinus disease Sinus secretione [120]
(MRSA) (www.aat-array.com/staphy.htm) [35] and the fastidiosum
Mycochip which detects 14 mycobacterial species. Qiagen, Haemophilus segnis Pyelonephritis Blood [121]
Inc. (Valencia, CA) offers arrays for many bacterial patho- Helicobacter pylori Dyspepsia Supragingival [122]
plaque
gens including Bacillus anthracis, C. jejuni, E. coli,
Neisseria meningitidis Meningitis csf [40,53]
Haemophilus influenzae, Neisseria spp., and Salmonella Staphylococcus Sinus disease Sinus secretione [120]
spp. The Nanochip (Nanogen, Inc., San Diego, CA) is one epidermis
example of a self-contained electronic microarray chip, Staphylococcus Onycholysis, Wound discharge [123]
primarily used for single nucleotide polymorphism (SNP) intermedius localized cellulitis
Streptococcus Thoracic empyema Pleural fluid [124]
detection in human genetic applications, but has recently
pneumoniae
been evaluated for bacterial detection. Designed to target long Streptococcus pyogenes Thoracic empyema Pleural fluid [124]
(350 base pair) and short (200 base pair) regions of the 16S a
Tissue, pleural fluid, blood, lymph node.
rRNA gene of 8 marine bacteria, the Nanochip demonstrated b
Tissue, blood.
variable specificity, and optimization was recommended c
Blood, sputum, abscess.
d
[36]. If a commercial microarray is not desirable, custom Typically found in feces of healthy individuals, this organism was
microbial diagnostic microarrays are available from or- associated with acute gangrenous appendicitis and septic shock in this case.
e
Serous, mucous, or pus.
ganizations like the Department of Bioresources at Seibers-
dorf, Austria and others. For these custom products,
oligonucleotide probes based on 16S rRNA, 16S – 23S pathogenic bacteria from clinical specimens that have been
inter-gene spacer, or other functional genes are designed identified by 16S rRNA sequencing.
(http://www.diagnostic-arrays.com/coop.htm). Non-com- Bacterial identification by 16S rRNA gene sequencing
mercial arrays for bacterial detection have also been has become increasingly popular with clinical laboratories
developed such as that described by Roth et al. Using broad since costs have decreased and improved throughput has
range primers targeting topoisomerases, a diagnostic array become available in the last few years [39]. One recent
was developed for the identification of 9 upper respiratory study even indicates that the cost for 16S rRNA gene
bacterial pathogens [37]. A clinical laboratory might also sequencing can be as low as a third the cost of con-
consider using a validated microarray for detection of ventional identification methods [46]. To minimize costs,
antimicrobial resistance genes. Recently, Yu et al. developed packages such as the MicroSeq 500 and the MicroSeq
and validated a diagnostic microarray for the detection of 16S rRNA gene systems (Applied Biosystems, Foster
fluoroquinolone-resistant E. coli clinical isolates [38]. City, CA) are commercially available [47,48]. For those
clinical laboratories that do not have sequencing capa-
3.3. Sequencing-based identification bilities, 16S rRNA gene sequencing services can be
outsourced.
If an assay for a specific bacterial pathogen is not The speed with which identification can be made by 16S
available or when multiple agents may be implicated as the rRNA gene sequencing compared to traditional biochemical
cause of disease, a broad-range detection approach can be methods is also an advantage [39,46,49,50] especially in
useful. Universal targets such as the 16S rRNA genes or the cases where conventional methods were inadequate [40].
16S – 23S rRNA gene interspacer region have been used For example, fastidious organisms, such as Mycobacteria
extensively for bacterial identification, especially if bacteria spp., can be identified in 24 h by 16S rRNA gene
are difficult to isolate by conventional methods [39 – 41]. sequencing [51]. In fact, for some pathogens, 16S rRNA
16S rRNA gene sequencing has also been used successfully gene sequencing may be the only rapid NAT available as
to detect pathogens from culture-negative specimens [42]. PCR-based tests have not yet been developed [46]. As with
Furthermore, recent work has indicated that 16S rRNA gene any diagnostic method, there are some pitfalls of using a
sequences may form the basis for sub-typing schemes for universal gene target such as 16S rRNA. Some of the
some pathogens [43 – 45]. Table 3 lists some of the sequences in the public databases (i.e. GenBank) are known
 E.A. Mothershed, A.M. Whitney / Clinica Chimica Acta 363 (2006) 206 – 220
to contain errors [52], especially those submitted prior to the
development of high-fidelity, automated sequencing sys-
tems [49,50,52]. Because 16S rRNA gene primers target
conserved sequences and the 16S rRNA gene is ubiquitous
among all bacteria, a sequence may be amplified from a
contaminating bacterium instead of the etiologic pathogen
[53]. Other universal targets such as heat-shock proteins,
like hsp65 [54] or cold-shock proteins [55] have also been
used to identify bacteria from clinical specimens.
Pyrosequencing (Pyrosequencing AB, Uppsala, Sweden)
is a technology whereby a single-stranded DNA template is
prepared, a sequencing primer is hybridized to a compli-
mentary sequence on the template, and enzymes catalyze a
light reaction when each nucleotide is incorporated into the
growing DNA strand [56]. Pyrosequencing has been used to
identify and characterize bacterial pathogens such as
Helicobacter pylori [57], N. meningitidis [58], and N.
gonorrhoeae [59], and rapidly discriminate pathogenic from
non-pathogenic bacteria in complex specimens [60]. The
major advantage of this technology is that the price per
sample reaction may cost 10-fold less than fluorescent-
based sequencing [61].
4. Detection of bacterial pathogens by nucleic acid
hybridization or mass spectrometry
The discovery of PCR has revolutionized molecular
diagnostics over the past decade, and it seems that the
number of PCR-based NATs being developed mimics the
exponential amplification of target molecules! However,
non-amplification methods for detecting bacteria are also
commercially available or are evolving to the point that they
can be easily performed in a clinical laboratory. Many of the
commercially available non-amplification NATs rely on
detection of a specific target by chemiluminescence,
colorimetric, or fluorescent signals.
4.1. Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) assays use
fluorescently labeled 16S rRNA or 23S rRNA probes and
fluorescent microscopy to detect intact bacteria directly in
clinical specimens, such as blood or tissue, or after
enrichment culture. FISH is a useful method for detection
of fastidious organisms such as Bartonella spp. and
Yersinia pestis. In fact, multiple species can be detected
simultaneously using two or more specific probes labeled
with unique fluorescent dyes. The procedure takes
between 1 and 2 h and consists of fixing the specimen,
preparing a smear or section on a microscope slide,
permeabilizing the cells, hybridizing the target sample
with the probe and detecting hybridization by fluorescence
microscopy.
Family-, genus-, and species-specific FISH probes have
been developed and published for detection of Chlamydia
213
spp. [62], Brachyspira spp. [63], Enterococcus spp.,
Pseudomonas aeruginosa [64], Helicobacter spp.[64],
Streptococcus spp. [65,66] Staphylococcus spp., Yersinia
spp., and others [67]. Many of these studies were aimed at
identifying bacteria in positive blood culture bottles obviat-
ing the need for subculture. A few FISH NATs are
commercially available including those from Microscreen
Ribotechnologies (Groningen, The Netherlands) for the
detection of human intestinal bacteria: Bifidobacterium
spp., E. coli, Lactobacillus spp., Streptococcus spp. and
Clostridia spp.
4.2. Peptide nucleic acid-FISH
In 1991, Nielson et al. discovered a DNA analogue called
peptide nucleic acid (PNA) [68]. Fluorescently labeled
PNAs have been successfully used as hybridization probes
in FISH assays. PNA probes have distinct advantages over
DNA probes including the stability of the PNA/RNA hybrid
due to the uncharged PNA [69]. Also, PNAs enter a
bacterial cell more easily because of their relative hydro-
phobicity. PNAs also have higher specificity than DNA
oligomers due to the higher Tm of the PNA probe compared
to its DNA counterpart [70].
AdvanDX (Woburn, MA) offers three PNA-FISH assays
for diagnostic use and all have gained FDA clearance.
AdvanDx PNA-FISH NATs for definitive identification of
S. aureus, Candida albicans and Enterococcus faecalis
directly from positive blood culture bottles are currently in
use in some US medical centers [70 –73]. In an independent
evaluation of the AdvanDX S. aureus PNA-FISH test,
Gonzalez et al. found that the test was 100% sensitive, 99%
specific, and gave 99% and 100%, positive and negative
predicative values, respectively, compared to three con-
firmatory tests [74]. For the detection of S. aureus, Chapin
and Musgnug performed a comparison of PNA-FISH
(AdvanDX), API RAPIDEC Staph System (bioMerieux),
and direct tube coagulase test, using the AccuProbe S.
aureus Culture Identification kit (Gen-Probe) as the stand-
ard. PNA-FISH demonstrated 99% sensitivity and 100%
specificity [75].
In addition to PNA-FISH, theoretically, PNAs could be
substituted for DNA oligonucleotides to enhance an assay’s
performance. PNAs have been incorporated into chemilu-
minescent in situ hybridization assays (CISH) [76,77],
microarrays [78], biosensors [79,80], PCR clamping assays
for mutant allele detection [81], Molecular Beacons [82] and
light-up probes for real time PCR [83].
4.3. Line probe assay
The line probe assay (LiPA) consists of a nitrocellulose
strip with specific oligonucleotide probes attached as
discreet parallel lines along the strip. Hybridization results
in a color change that can be detected visually or by an
automated reader. Innogenetics (Gent, Belgium) produces
214 E.A. Mothershed, A.M. Whitney / Clinica Chimica Acta 363 (2006) 206 – 220

several line probe NATs for bacterial detection including
ones for M. tuberculosis complex and Mycobacterium spp.,
rpoB gene mutations conferring rifampicin resistance, and
Treponema pallidum antibodies. The INNO-LiPA Rif.TB
test detects the M. tuberculosis complex, specifically five
genotypes corresponding to sensitivity to rifampicin and
four resistant genotypes. Test results were 100% concordant
with antibiogram results [84], and in a separate study, the
sensitivity and specificity of this test were 100% and 92%,
respectively [85]. The INNO-LiPA MYCOBACTERIA v2
differentiates 16 mycobacterial species using probes specific
for the 16S – 23S rRNA spacer region. The first version of
this test was evaluated in 2000 and correctly identified 50 of
53 isolates to the species level [86]. The test was recently
improved and evaluated with 642 Mycobacterium spp.
isolates and 27 non-mycobacterial isolates [87] and dem-
onstrated 100% sensitivity and specificity and 99.2%
accuracy [87].
4.4. Hybridization protection assay
Hybridization protection assays (HPA) utilize a chemilu-
minescent acridinium ester detector molecule on a DNA
probe that targets the specific bacterial rRNA. The RNA/
DNA hybrid is detected in a luminometer. AccuProbe (Gen-
Probe, San Diego, CA) HPA tests are available for the
detection of M. avium, M. avium complex, M. intracellulare,
M. gordonae, M. kansasii, M. tuberculosis complex,
Campylobacter spp., Enterococcus spp., Group A Strepto-
coccus (S. pyogenes), Group B Streptococcus (S. agalactia),
H. influenzae, N. gonorrhoeae, S. aureus, S. pneumoniae and
L. monocytogenes. After investigators adjusted the cutoffs for
a positive result to increase the sensitivity of the S. aureus, S.
pneumoniae, Enterococcus spp., Group A Streptococcus
and Group B Streptococcus tests, four had > 90%
sensitivity, >98% specificity, > 94% positive predictive
value, and >95% negative predictive value. The S. aureus
test, however, demonstrated lower sensitivity (81%) [88].
4.5. Mass spectrometry
Mass spectrometry (MS) causes ionization and disinte-
gration of a target molecule by bombarding it with electrons.
The mass/charge ratio of the resulting molecular fragments is
then analyzed to produce a molecular signature. MS has
often been used to identify bacteria by protein signature but
was not considered a useful tool for DNA studies. However,
in the past 15 years the difficulties of analyzing DNA have
been largely overcome [89], and the use of MS for nucleic
acid analysis has developed rapidly. Matrix-assisted laser
desorption/ionization time of flight (MALDI-TOF) MS
generates sample data in seconds. Because of its rapidity
and the capability of analyzing thousands of samples per day,
many researchers are now investigating the use of MS as a
diagnostic tool for bacterial detection. MALDI-TOF MS has
proven to be valuable for SNP detection in human DNA, and
was recently used to differentiate virulent N. meningitidis
clones [90]. An SNP within the fumC gene was used to
discriminate between the hypervirulent ET-15 strain and
other ET-37 complex strains, and MALDI-TOF proved to be
an efficient alternative to traditional DNA sequencing [90].
Analysis of uncultured bacteria is also possible by MALDI-
TOF MS. By incorporating dUTP rather than dTTP during
16S rRNA gene PCR, researchers were able to detect single
nucleotide differences in fragment patterns after uracil –
DNA –glycosylase treatment [91]. Lefmann et al. were able
to genotype 12 type strains and 24 clinical isolates of
Mycobacteria using RNA transcripts of 16S rRNA genes
[92]. The data published in the last two years on bacterial
DNA analysis, and the TIGER project (see Section 7) may
compel more research in MALDI-TOF MS for clinical
diagnostics.

Table 4
Instruments for automated nucleic acid extraction
Manufacturer Instrument Model Capture method Maximum Maximum Elution Processing time for Approximate
no. of samples sample volume volume maximum samples cost
Applied Biosystems ABI Prism 6100 Silica/filtration 96 700 Al 150 Al 0.5 h $150,000
6700 Silica/filtration 96 700 Al 40 – 200 Al 1.5 h $154,500
Autogen AutoGenPrep 245 Centrifugation 24 Tissue Pellet 3.5 h Inquirea
AutoGenPrep 965 Centrifugation 384 200 Al Pellet 4–6 h Inquire
AutoGenFlex 3000 Centrifugation 40 5 mL Pellet 5h Inquire
bioMerieux NucliSens Extractor Silica/filtration 10 2 mL 35 Al 45 min $80,000
Mini-Mag Silica/magnetic 12 1 mL 25 Al <1 h $12,000
Qiagen BioRobot EZ-1 Silica/magnetic 6 350 Al 200 Al 0.33 h $29,000
M48 Silica/magnetic 48 350 Al 100 – 400 Al 3.5 h $73,450
M96 Silica/magnetic 96 50 Al 50 – 100 Al 3.5 h $93,150
MDx Silica/filtration 96 200 Al 200 Al 2.5 – 4 h $170,000
9604 Silica/filtration 96 200 Al 400 Al 2h $70,000
Roche MagNA Pure LC Magnetic glass particles 32 150 Al 100 Al 1 – 1.5 h $84,500
Compact Magnetic glass particles 8 1000 Al 50 – 200 Al 35 min $35,000
a
www.autogen.com.
 E.A. Mothershed, A.M. Whitney / Clinica Chimica Acta 363 (2006) 206 – 220 215

5. Quality control: nucleic acid extraction and Table 6

contamination prevention
When choosing to use a NAT, it is important to recall the
adage ‘‘Garbage in, garbage out’’. In other words, results
produced by the NAT are only as good as the quality of the
nucleic acid that goes into the test. The presence of
inhibitors in clinical specimens, for example, may interfere
with PCR or hybridization reactions and cause false
negative results [93]. Therefore, selection of the nucleic
acid extraction method is of critical importance. Many
commercially available extraction kits incorporate a buffer
to lyse the bacteria and a silica matrix membrane (typically
in column format) to trap the DNA or RNA. Several wash
steps are required to remove protein and other macro-
molecules, and the purified DNA and RNA is then eluted
from the membrane. Many of the manual extraction
methods require several centrifugation steps. To reduce
hands-on time, operator error, and sample contamination,
semi-automated DNA or RNA extraction kits and equip-
ment have been designed and are commercially available
(Table 4).
Strategies to prevent contamination should also be
considered when using NATs, as the inherent sensitivity of
these assays makes detection of undesirable nucleic acid a
potential problem [94,95]. Some simple contamination
control methods are listed in Table 5 and were recently
reviewed in detail by Aslanzadeh [96] and Borst et al. [95].
Importantly, it should be noted that each assay mentioned
in this review may have deficiencies such as suboptimal
sensitivity, specificity or efficiency. It is not the intent of this
review to recommend one assay or instrument over another.
It is the responsibility of the clinical laboratory to conduct
its own validation of any assay or procedure before
implementing it for routine diagnostic use [97,98].
6. How to choose which test is right for your laboratory?
With the abundance of tests and technologies available, a
laboratory must consider what factors are most important for
achieving the desired results before choosing a NAT. The
first question should be does a need exist for NATs or are
Criteria to consider when choosing a nucleic acid-based test
Criterium Test or method to consider
High-throughput Real-time PCR (96 or 384 well assays);
multiplex real-time PCR; automation
Low limit of detection NAATs
Multiple target detection Multiplex PCR; multiplex real-time
PCR; microarray
Broad-based bacterial detection 16S rRNA or 23S rRNA gene
sequencing
Antimicrobial resistance Real-time PCR; microarray;
detection commercial NATs
Ease of use Commercial NATs
traditional methods adequate to produce timely, reliable
results? While many factors must be considered before
implementing NATs, including cost assessment, some
important points to consider as listed in Table 6.
7. Future trends for NATs
Certainly, a rapid, simple test that provides an accurate
diagnosis is the ‘‘holy grail’’ of any clinical laboratory or
clinician. While technology may not yet allow for such a
reality, it is intriguing to consider the advances being made
in molecular diagnostics and perhaps glimpse that such a
goal is not far out of reach. Some innovative technologies
and future applications that are currently in development
and evaluation are shown in Table 7.
Notably, miniature devices, known as biosensors, are
being developed for chemical and microorganism detec-
tion. In general terms, a biosensor is an instrument that
detects a target (analyte) by capturing the target (hybrid-
ization or binding to antibody) and transducing the capture
event into a detectable signal. Many of the sensors in
development use DNA or protein as the sensor and an
optical, acoustic, surface plasmon resonance (SPR), micro-
gravimetric, chemiluminescent, fluorescent, or electro-
chemical signal output (Table 7). These devices have
been of keen scientific interest for detection of bacteria
because they have the potential to be fast, reliable,
sensitive, and mass-produced. In particular, biosensors
have been a priority for blood collection centers to detect

Table 5
Quality control strategies to consider when performing nucleic acid-based tests
Strategy
Designate a ‘‘clean’’ area for reaction set-up
Use personal protective equipment (PPE)
Use uracil-N-glycosylase (UNG) in real-time
PCR reactions
Use a ‘‘hot-start’’ method
Use controls in every test
How it works
Room under negative air pressure; positive-displacement pipettes; aerosol-block pipette tips; UV-equipped
PCR cabinet
Disposable gloves and lab coats should be worn to prevent introduction of contaminating DNAs or
nucleases
Real-time PCR incorporates dUTP not dTTP during amplification; UNG cleaves DNA containing UTP,
thus destroying contaminating carry-over amplicons
Minimizes false priming events by withholding a crucial reaction component until appropriate temperature
is reached
External positive and negative controls monitor reaction performance and contamination; internal controls
monitor presence of inhibitors
216 E.A. Mothershed, A.M. Whitney / Clinica Chimica Acta 363 (2006) 206 – 220

Table 7
Innovative technologies for bacterial pathogen detection
Device Manufacturer Detection method URL or reference
RAPID, Ruggedized Advanced Pathogen Idaho Technology, Salt Lake City, UT Real-time PCR http://www.idahotech.com/
Identification Device
RAZOR Idaho Technology, Salt Lake City, UT Real-time PCR http://www.idahotech.com/
HANAA, Handheld Advanced Nucleic Lawrence Livermore National Laboratory, Real-time PCR [125]
Acid Analyzer Livermore, CA
Bio-Seeq Smiths Detection, Pinebrook, NJ Real-time PCR [126]
DiversiLab Bacterial Barcodes, Inc., Houston, TX rep-PCR [127 – 129]
LIAT Lab-in-a-tube Iquum, Inc., Allston, MA Various http://www.iquum.com
GeneXpert Cepheid, Sunnyvale, CA Real-time PCR http://www.cepheid.com
Unnamed (microfluidic) HandyLab, Ann Arbor, MI Various http://www.handylab.com
Nanochip Nanogen, San Diego, CA Various http://www.nanogen.com
Quantum Dot Quantum Dot, Hayward, CA Nanocrystals [130]
Verigene Nanosphere, Northbrook, IL Gold nanoparticles http://www.nanosphere-inc.com
Scansystem Hemosystem, Marseilles, France Chemiluminescent [131,132]
Biacore Biacore, Piscataway, NJ Optical [133 – 135]
Spreeta Texas Instruments, Attleboro, MA Optical [133 – 135]
Tiger Isis Pharmaceuticals Carlsbad, CA and Science
Applications International
Corporation, San Diego, CA Mass spectrometry [136]
Unnamed Not commercially available Magnetic field [137]
Unnamed Not commercially available Microbalance [138,139]
Unnamed Not commercially available Acoustic [140]
Unnamed Not commercially available Electrochemical [141,142]
Unnamed Not commercially available Amperometric [143]
Unnamed Not commercially available Optical [144]
Unnamed Not commercially available NASBA [145]

blood product contamination and for national security
agencies to detect biowarfare agents in air, food and water.
Many of these instruments could potentially become part of
a clinical laboratory or even used as portable devices for
bedside diagnostic use.
8. Conclusions
There is great potential of molecular assays to increase
the speed and accuracy of bacterial identification in the
clinical laboratory. The limitations of NATs must be
considered, however, when deciding whether to incorporate
these tests in the diagnostic algorithm. False positive and
false negative results may have a significant impact on
patient management [95]. To reduce such errors, contami-
nation must be minimized, technicians must have proper
training, and quality control procedures must be incorpo-
rated into routine laboratory workflow. While the cost of
some molecular diagnostic instruments is high, the benefits
of faster turnaround time, high throughput, and enhanced
sensitivity over traditional methods may override this
obstacle. Another important consideration is that laboratory
space must be allocated for instruments and dedicated as
DNA-free areas.
It is purported often that the new molecular methods can
be sensitive and specific, but what does a ‘‘positive’’ test
result mean clinically? Caution must be taken when
interpreting NATs results. A positive result for Bartonella
in a patient with culture-negative endocarditis is a possible
true positive, but what if the result from that patient’s
specimen yielded Burkholderia picketti? Researchers have
shown that DNA from microorganisms can be amplified
from plastic ware, PCR reagents, the lab environment, and
water (reviewed in [95]). Nikkari et al. amplified bacterial
DNA from healthy individuals’ blood [99].
Negative results will also pose a challenge for the
clinician. When interpreting a negative result, consideration
must be given to the specimen type, antibiotic treatment of
the patient, and bacterial concentration in the small sample
volume used for the NAT. Therefore, all molecular and
immunological diagnostic results must be reviewed and
critically interpreted based on the patient’s clinical presen-
tation before definitive diagnoses and treatment decisions
are made. It is not the intention of this review to recommend
using NATs as first line diagnostic tools, but rather to
emphasize the important role of these tests in assembling an
accurate and timely diagnosis of bacterial disease.
Molecular diagnostic methods will most likely continue
to be incorporated into clinical laboratory work as more
commercialized assays are developed and cleared through
FDA, prices decrease due to evolving technology, and
clinical microbiologists and clinicians become accustomed
to the advantages of NATs. The limitations of these
systems will continue to be solved. Integrated, closed
microsystems will help in contamination prevention, and
assays will become more standardized and easier to
perform. With time, integrated NATs for bacterial detection
may be developed for hospital bedside, doctors’ offices or
even home use.
 E.A. Mothershed, A.M. Whitney / Clinica Chimica Acta 363 (2006) 206 – 220
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