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Bioelectronic DNA detection of human papillomaviruses using eSensor™: a model system for detection of multiple pathogens

  • Suzanne D Vernon1Email author,
  • Daniel H Farkas2, 3,
  • Elizabeth R Unger1,
  • Vivian Chan2, 4,
  • Donna L Miller1,
  • Yin-Peng Chen2,
  • Gary F Blackburn2 and
  • William C Reeves1
BMC Infectious Diseases20033:12

DOI: 10.1186/1471-2334-3-12

Received: 06 January 2003

Accepted: 19 June 2003

Published: 19 June 2003

Abstract

Background

We used human papillomaviruses (HPV) as a model system to evaluate the utility of a nucleic acid, hybridization-based bioelectronic DNA detection platform (eSensor™) in identifying multiple pathogens.

Methods

Two chips were spotted with capture probes consisting of DNA oligonucleotide sequences specific for HPV types. Electrically conductive signal probes were synthesized to be complementary to a distinct region of the amplified HPV target DNA. A portion of the HPV L1 region that was amplified by using consensus primers served as target DNA. The amplified target was mixed with a cocktail of signal probes and added to a cartridge containing a DNA chip to allow for hybridization with complementary capture probes.

Results

Two bioelectric chips were designed and successfully detected 86% of the HPV types contained in clinical samples.

Conclusions

This model system demonstrates the potential of the eSensor platform for rapid and integrated detection of multiple pathogens.

Background

Global emergence of pathogenic infectious diseases by both natural and intentional means presents a formidable challenge to infectious disease surveillance and response, namely timely and efficient pathogen detection. Many laboratory methods exist for identifying pathogens, but most require exquisite care in sample handling and processing prior to characterization of a pathogen. In addition, costly and perishable reagents, equipment, and supplies are required for sensitive and specific detection. The ideal detection system would integrate sample processing and pathogen characterization into a single automated device that would eliminate laborious, and time consuming sample processing and costly detection. Bioelectronic detection of nucleic acids on a miniature solid support is one of the first steps toward development of such an integrated detection device.

Bioelectronic DNA detection involves forming an electronic circuit mediated by nucleic acid hybridization and it serves as the basis for a DNA detection system called eSensor™ [14]. This system uses low-density DNA chips containing electrodes coated with DNA capture probes. Target DNA present in the sample hybridizes specifically both to capture probes and ferrocene labeled signal probes in solution thereby generating an electric current. Current eSensor DNA chips contain as many as 36 electrodes for simultaneous detection of multiple pathogens from a single sample.

Many pathogens cause both acute and chronic disease at relatively low copy number and may be difficult or impossible to propagate in culture. Thus, most pathogen detection systems rely on nucleic acid amplification by using polymerase chain reaction (PCR). One highly effective amplification strategy targets conserved sequences among the family of organisms of interest. Such broad-range PCR strategies have been used to identify and characterize several known and previously uncharacterized bacteria [5, 6] and viruses [7, 8]. In order to maximize the utility of these effective pathogen nucleic acid amplification systems, amplification needs to be coupled with rapid, sensitive, and specific detection. Bioelectronic DNA detection by use of the eSensor chip might fulfill this need.

Human papillomaviruses (HPV) serve as an ideal model system for determining the efficiency and feasibility of eSensor DNA detection technology since there are at least 30 distinct genital HPV types that can be effectively amplified with broad-range consensus PCR primers [9]. We designed two eSensor chips, each containing 14 probes specific for the conserved L1 region of the HPV genome. We evaluated clinical cervical cytology samples known to contain one or more HPV types. The eSensor DNA detection platform successfully detected the correct HPV type in most of these clinical samples, demonstrating that the system provides a rapid, sensitive, specific, and economical approach for multiple-pathogen detection and identification from a single sample.

Methods

eSensor™ DNA Chip Design

We used previously described printed circuit board technology to manufacture eSensor chips with 16 gold electrodes, one reference electrode, and one auxiliary electrode [24]. Each electrode was wired to a connector at the edge of the chip. Capture probes were synthesized using an alkane chain of 16 residues terminated at the 3'-end with a disulfide which was spotted onto the gold electrode on the chips to form a self-assembled monolayer [10]. The chips were glued into a plastic housing with a port for sample introduction.

Hybsimulator (RNAture, Inc., Irvine, CA) was used to design the HPV target mimics, and signal and capture probes. For these experiments, we designed capture (Table 1) and signal (Table 2) probes to hybridize specifically to the exon sequence of β-globin and to HPV sequences within the 450-bp L1 region amplified by the PGMY primer set. Signal probes (Table 2) tagged with eight ferrocenes were synthesized with a modified adenine residue (N6) containing a ferrocene substitution on the ribose ring [11]. No signal probes were synthesized for HPV types 33, 39, 55, and 73. HPV target mimic oligonucleotides were synthesized for types 6, 11, 16, 18, 26, 31, 35, 40, 42, 45, 51, 52, 54, 56, 58, 59, 66, 68, 82, 83, and 84 (Table 3).
Table 1

Human papillomavirus (HPV) capture probe and PCR primers sequences.

6

capture

CAG AAT TGG TGT ATG TGG AAG A(N152)

11

capture

TAA TCT GAA TTA GTG TAT GTA GCA GAT TTA GAC A(N152)

16

capture

GTA GTT TCT GAA GTA GAT ATG G(N152)

18

capture

TGG TAG CAT CAT ATT GCC CAG G(N152)

26

capture

ATC AGA TGG TTT AAA TGG AGT GGA TGC(N152)

31

capture

TAC TAC TTT TAA ATG TAG TAT CAC(N152)

35

capture

ACT GTC ACT AGA AGA CAC AGC AGA ACA CA(N152)

40

capture

GGG GGA CTG TGT GGC ACC A(N152)

42

capture

AGC AGC TGT ATA TGT ATC ACC AGA TGT TGC AGT GGC TCA(N152)

45

capture

CTT AGT AGG GTC ATA TGT ACT TGG C(N152)

51

capture

TTG GGG AAA CCG CAG CAG TGG CAG GGC TA(N152)

52

capture

TAT GTG CTT TCC TTT TTA ACC T(N152)

54

capture

GTC AGA ATT ATT AAA GCT ATC CTG CG(N152)

56

capture

TTT TCG TGC ATC ATA TTT ACT TA(N152)

58

capture

GTA CCT TCC TTA GTT ACT TCA G(N152)

59

capture

CTG GTA GGT GTG TAT ACA TTA G(N152)

66

capture

CAC GGG CAT CAT ATT TAG TTA A(N152)

68

capture

TTA AAT TTA TTA GGA TCA TAA ATA TTT GGT A(N152)

82

capture

AAT GTT TGT GCA ACA GAT TGA GTA ACA GCT GTG(N152)

83

capture

GTT AGA GGC TGT GTA TTC ATT AGC(N152)

84

capture

GGT TTA TAT TCT GAT TCG GTG T(N152)

globin

capture

AGC AAT AGA TGG CTC TGC CC(N152)

globin

primer

GAA GAG CCA AGG ACA GGT AC

globin rev

primer

BIOTIN CAA CTT CAT CCA CGT TCA CC

HMB01 rev

primer

BIOTIN GCG ACC CAA TGC AAA TTG GT

PGMY09F

primer

BIOTIN CGT CCC AAA GGA AAC TGA TC

PGMY09G

primer

BIOTIN CGA CCT AAA GGA AAC TGA TC

PGMY09H

primer

BIOTIN CGT CCA AAA GGA AAC TGA TC

PGMY09I

primer

BIOTIN GCC AAG GGG AAA CTG ATC

PGMY09J

primer

BIOTIN CGT CCC AAA GGA TAC TGA TC

PGMY09K

primer

BIOTIN CGT CCA AGG GGA TAC TGA TC

PGMY09L

primer

BIOTIN CGA CCT AAA GGG AAT TGA TC

PGMY09M

primer

BIOTIN CGA CCT AGT GGA AAT TGA TC

PGMY09N

primer

BIOTIN CGA CCA AGG GGA TAT TGA TC

PGMY09P

primer

BIOTIN GCC CAA CGG AAA CTG ATC

PGMY09Q

primer

BIOTIN CGA CCC AAG GGA AAC TGG TC

PGMY09R

primer

BIOTIN CGT CCT AAA GGA AAC TGG TC

PGMY11A

primer

GCA CAG GGA CAT AAC AAT GG

PGMY11B

primer

GCG CAG GGC CAC AAT AAT GG

PGMY11C

primer

GCA CAG GGA CAT AAT AAT GG

PGMY11D

primer

GCC CAG GGC CAC AAC AAT GG

PGMY11F

primer

GCT CAG GGT TTA AAC AAT GG

Table 2

HPV type and signal probe sequence.

6

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) TGT AGT TAC GGA TGC AC

11

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) CAG ATG CAG ATA GTG TCA T

16

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) CAG CGC ATA ATG ACA TAT TT

18

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) TAC AGG AGA CTG TGT AG

26

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) AGA TGC TGT AGA TAA TGT AC

31

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) TGT TTG CAA TTG CAG CA

35

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) CAG ACA TAT TTG TTC TAC GG

40

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) TAA GGT TAA ATT AGT GCA ACG A

42

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) CAA AGA CAT GTT AGT ACT AC

45

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) ACA GGA TTT TGT GTA GAG G

51

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) ATA GTT AAA TTT GTA CTT CTG G

52

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) CAG CAC ATA AAG TCA TG

54

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) TGG ATG CTG TAG CAC AC

56

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) ACT GTT CTG TAG CAG TAC T

58

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) TGC ATA ATG TCA TAT TAG TG

59

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) GAA TAG AAG AAG TAG TAG AA

66

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) TGT GCT TTT AGC TGC AT

68

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) CAG CTG ATT CAG TAG TAG TA

82

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) CTA ATG GTT AAA TTG GTA GTT

83

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) CTG TGT AGC AGG AGC TGA AA

84

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) TGG TAG CAG CAC TAA TA

Globin

(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) TGA CTT TTA TGC CCA GAC CTG G

Table 3

HPV type and target mimic oligonucleotide sequences

6

ACC ACA CGC AGT ACC AAC ATG ACA TTA TGT GCA TCC GTA ACT ACA TCT TCC ACA TAC ACC AAT TCT GAT TAT AAA

11

GTA CAA ATA TGA CAC TAT GTG CAT CTG TGT CTA AAT CTG CTA CAT ACA CTA ATT CAG ATT ATA AGG AAT ACA TGC G

16

ACT ACA CGC AGT ACA AAT ATG TCA TTA TGT GCT GCC ATA TCT ACT TCA GAA ACT ACA TAT AAA AAT ACT AAC TTT AA

18

ACC ACT CCC AGT ACC AAT TTA ACA ATA TGT GCT TCT ACA CAG TCT CCT GTA CCT GGG CAA TAT GAT GCT ACC AAA T

26

GTA CTA ACC TTA CCA TTA GTA CAT TAT CTG CAG CAT CTG CAT CCA CTC CAT TTA AAC CAT CTG ATT ATA AAC AAT T

31

ACC ACA CGT AGT ACC AAT ATG TCT GTT TGT GCT GCA ATT GCA AAC AGT GAT ACT ACA TTT AAA AGT AGT AAT TTT AA

35

TGT AGT TGA TAC AAC CCG TAG TAC AAA TAT GTC TGT GTG TTC TGC TGT GTC TTC TAG TGA CAG TAC ATA TAA AAA T

40

AGT TGT AGA CAC CAC TCG TAG CAC TAA TTT AAC CTT ATG TGC TGC CAC ACA GTC CCC CAC ACC AAC CCC ATA TAA T

42

ACT ACC CGT AGT ACT AAC ATG ACT TTG TGT GCC ACT GCA ACA TCT GGT GAT ACA TAT ACA GCT GCT AAT TTT AAG G

45

AAC ATT ATG TGC CTC TAC ACA AAA TCC TGT GCC AAG TAC ATA TGA CCC TAC TAA GTT TAA GCA GTA TAG TAG ACA T

51

GTT GAT ACT ACC AGA AGT ACA AAT TTA ACT ATT AGC ACT GCC ACT GCT GCG GTT TCC CCA ACA TTT ACT CCA AGT A

52

ACC ACT CGT AGC ACT AAC ATG ACT TTA TGT GCT GAG GTT AAA AAG GAA AGC ACA TAT AAA AAT GAA AAT TTT AAG

54

GTA CTA ACC TAA CAT TGT GTG CTA CAG CAT CCA CGC AGG ATA GCT TTA ATA ATT CTG ACT TTA GGG AGT ATA TTA G

56

GTA CTA ACA TGA CTA TTA GTA CTG CTA CAG AAC AGT TAA GTA AAT ATG ATG CAC GAA AAA TTA ATC AGT ACC TTA G

58

ACC ACT CGT AGC ACT AAT ATG ACA TTA TGC ACT GAA GTA ACT AAG GAA GGT ACA TAT AAA AAT GAT AAT TTT AAG

59

ACT ACT CGC AGC ACC AAT CTT TCT GTG TGT GCT TCT ACT ACT TCT TCT ATT CCT AAT GTA TAC ACA CCT ACC AGT

66

ACT ACC AGA AGC ACC AAC ATG ACT ATT AAT GCA GCT AAA AGC ACA TTA ACT AAA TAT GAT GCC CGT GAA ATC AAT

68

GTA CCA ATT TTA CTT TGT CTA CTA CTA CTG AAT CAG CTG TAC CAA ATA TTT ATG ATC CTA ATA AAT TTA AGG AAT A

82

ACT ACT AGA AGT ACC AAT TTA ACC ATT AGC ACT GCT GTT ACT CAA TCT GTT GCA CAA ACA TTT ACT CCA GCA AAC T

83

GTA CCA ATA TTA CTA TTT CAG CTG CTG CTA CAC AGG CTA ATG AAT ACA CAG CCT CTA ACT TTA AGG AAT ACC TCC G

84

ACC ACC CGC AGC ACC AAT TTT ACT ATT AGT GCT GCT ACC AAC ACC GAA TCA GAA TAT AAA CCT ACC AAT TTT AAG

globin

GGG AGG GCA GGA GCC AGG GCT GGG CAT AAA AGT CAG GGC AGA GCC ATC TAT TGC TTA CAT TTG CTT CTG ACA CAA C

Sample selection and DNA extraction

Residual ThinPrep (Cytyc Corporation, Boxborough, MA) cervical samples previously tested for HPV using the Roche line blot assay [9] were used in this study. Twenty samples containing 24 of the 27 HPV types on the reverse line blot were selected for extraction and testing. (No samples were positive for HPV 53, 55, and 57.) Therefore a total of 21 HPV types could be evaluated (there were 22 HPV capture and signal probes minus no clinical sample containing HPV 53 leaves 21 possible HPV type answers). Samples were coded so that investigators were blinded to the presence of multiple HPV types. DNA was extracted from 250 μL aliquots by using the QIAmp DNA Mini Kit (QIAGEN Inc., Valencia, CA). The procedure provided with the kit was followed except that the proteinase K digestion was done overnight at 56°C. Following DNA elution from the column, DNA was concentrated in a Centricon Centrifugal Filter Device YM-100 (Millipore Corporation, Bedford MA) according to manufacturer's specifications. Each DNA sample was brought to 50 μL with the addition of ultra pure water.

HPV PCR and Typing

Five microliters of purified DNA from cytology samples was used in each PCR assay. HPV DNA was amplified with the PGMY09/PGMY11 primer system that uses a multiplex format to amplify both HPV and β-globin in the same tube [9]. To allow for strand separation, only the downstream primers designated PGMY11 and one β-globin primer were biotinylated (Table 1). The use of one biotinylated primer to amplify the product was still sufficient to verify the amplified HPV type by reverse line blot. Amplification without DNA template was used to monitor contamination.

Avidin-coated beads were used to harvest the biotin-labeled DNA strand from 50 μl of product. The biotin-free DNA strand was then used in the assay. Briefly, the target mimic or target DNA was mixed with a signal probe cocktail (125 nM of each signal probe) in a hybridization buffer (proprietary). This mixture was injected into the eSensor chip for hybridization at 40°C for 2 to 8 hours. The chips were then scanned by an AC voltammetry technique built into the eSensor detection device called the eSensor 4800, from -50 to +550 millivolts, to detect and interpret the electrochemical signal generated during hybridization. Investigators recording eSensor results remained blinded to prior or current HPV results.

Forty microliters of the amplified products was also independently tested for HPV using a reverse line probe assay (typing strip and reagents generously provided by Janet Kornegay, Roche Molecular Systems, Inc., Alameda, CA). In each case the HPV results on the strip were concordant with prior results. HPV detection and typing results from eSensor and Roche were then directly compared.

Results

The electrochemical signal is generated when the HPV amplicon hybridizes to the capture probe and ferrocene-labeled signal probe, thereby bringing the reporter molecule, ferrocene, into contact with the self-assembled monolayer on the gold electrode (Fig. 1). An alternating current voltammogram is obtained when HPV is detected in a sample but no electronic signal is registered when HPV DNA is absent from the sample. (Fig. 2).
Figure 1

Schematic represention of the eSensor DNA Detection System. A self-assembled monolayer is generated on a gold electrode. For electrochemical detection to occur across the monolayer, two hybridization events must occur: the first between the capture probe (shown in black perpendicular to the electrode) and the target (for example, HPV DNA, shown in gray), and the second between an adjacent region of the target and ferrocene labeled signal probe (shown in black parallel to the monolayer).

Figure 2

The eSensor™ bioelectronic representative signal output. A fourth harmonic alternating current voltammogram is obtained from an HPV-positive sample (left panel), and one obtained from an HPV-negative sample (right panel). The faradic signal in the fourth harmonic closely fits the third derivative of a Gaussian curve. The peak current (100 nA) in the fourth harmonic voltammogram is determined by averaging the two peaks that center around 130 mV.

Many of the HPV target mimics tested resulted in a specific signal, with the exception of HPV types 16, 26, 35, 40, 42, 51, 52, and 82. This nonspecific hybridization signal generally resulted from a long stretch of complementary sequences (6–8 bases) among the HPV types due to their close genetic relatedness. Nonspecific hybridization was eliminated by introducing a mismatched base to the complementary region in the signal probes for HPV types 16, 26, 35, 42, and 82 and the capture probes for HPV types 40, 42, 51, 52, and 82. For instance, the HPV 16 signal probe hybridized with both the HPV 16 target mimic (Fig. 3, left panel) and the HPV 52 capture probe (Fig. 3, center panel). This cross-reactivity was eliminated by changing the second A base in the 5'-end of signal probe to a G base (Fig. 3, right panel). In addition, we demonstrated that one base modification of the signal probe (Fig. 3, left and right panels) generated minimal negative impact to the assay signal level. Applying this strategy, we were able to effectively reduce the cross-reactivity among the signal and capture probes for those HPV types where problems of non-specific signal had initially been encountered without compromising hybridization strengths.
Figure 3

HPV 16 Signaling Probe Optimization. The HPV eSensor™ chip was decorated with 11 different HPV capture probes and an HIV capture probe as a negative control (shown in x-axis). An HPV 16 target mimic was detected by the eSensor in the presence of HPV 16 specific signaling probe (left panel) in which the sample also reacted with HPV 52-specific capture probe. The signal was identified as being from the hybridization between HPV 16 signal probe and the HPV 52 capture probe as shown in the mid-panel in which HPV 16 signal probe was introduced into the chip without any target mimic. The HPV 16 signal probe was modified replacing the second A base in the 5'-end with a G base. The resulted new signal probe hybridized specifically with the HPV 16 target mimic and did not cross-react with the HPV 52 capture probe.

In most instances for the 20 samples tested, there was perfect concordance between the HPV type(s) detected by reverse line blot and the eSensor system (Table 4). The eSensor detected 18 (86%) of 21 HPV types. In samples with multiple HPV types amplified by the consensus primer system and typed by reverse line blot (but blinded to the individual conducting the eSensor assay), the eSensor successfully detected the multiple HPV targets in all six samples (Table 4).
Table 4

Comparison of HPV detection and typing results

Target

HPV Type(s) by Reverse Line Probe

HPV Type(s) by eSensor™

Comments on eSensor™

6

6

6

 

11

11, 35, 40

11, 35

HPV 40 ND1

16

16

16

 

18

18

18

 

26

18, 26, 42

18, 26

HPV 42 ND

31

31

31, 84

weak 84 signal

35

35

35

 

40

11, 35, 40

11, 35

HPV 40 ND

42

42, 68

42, 68

 

45

45

ND

no globin signal

51

51

51

 

52

52

52

 

54

weak 54

ND

HPV 54 ND

56

56

56

 

58

58

58

 

59

59

59

 

66

66

66

 

68

42, 68

42, 68

 

82

82, 31, weak 51

82, 31

HPV 51 ND

83

83

83

 

84

84

84

 

1 ND, not detected

Discussion

We have presented a model application of a novel electronic detection platform [14]. We used HPV as a model infectious disease system because consensus primers are available that amplify at least 30 closely related genital HPV types. The resulting amplicons are extremely similar, and this relatedness challenges the ability of the eSensor technology to distinguish accurately among these pathogens. The first optimization steps involved evaluation of electrochemical signal generation by using HPV target mimics. Cross-hybridization was observed between a subset of HPV target mimics and the capture and signal probes. This cross-hybridization was easily overcome by the introduction of mismatched base pairs. The effectiveness of single-base mismatch discrimination of the eSensor has been demonstrated by using wild type and mutant strains of human immunodeficiency virus [3].

We used the eSensor DNA chip to determine the presence of single or multiple HPV types in cervical cytology samples. While the presence of the target HPV type was known, the presence of other HPV types in the same sample remained blinded (e.g., the HPV 42 in a clinical sample was identified but the presence of HPV 68 in the same sample was not known to those performing electrochemical detection). There was good concordance for the detection of the target HPV type by the eSensor DNA Detection System. Eighteen (86%) of 21 target HPV types represented on the chip were readily detected by the eSensor (Table 4); HPV 40, 45 and 54 were not detected. Some of the other discrepancies could be due to lack of rigorous optimization of sensitivity parameters for the eSensor system, such as the weak HPV 54 result on reverse line blot and lack of detection on eSensor. Low-level signals on the line probe assay have been validated by sequencing the amplified product [12]. For HPV 40 discrepancy, the capture probe design may not have been optimal to allow for hybridization with the HPV 40 target. These HPV types may be variant types that did not hybridize to the capture probe and subsequently could not be detected by the signal probe. Not only are there over 200 HPV types, but several sequence variants of HPV types are known to exist [13]. Most hybridization reactions could be analyzed after 2 hours, although in several instances up to 8 hours of hybridization time was required. The intent of this study was to use HPV as a model system to assess the feasibility of electrochemical detection with the eSensor chip of multiple pathogens, not to achieve perfect concordance of HPV detection with the reverse line blot.

In this model assay, we have performed optimization of probe sequences and assay protocol by using synthesized target mimics. The resultant assay correctly identified nearly all HPV type in the clinical samples. No attempt to further optimize the assay using PCR amplicons, rather than target mimics, was made and neither was the PCR process optimized for detection by using the eSensor DNA detection system. This work demonstrates the ease with which a multi-analyte panel assay can be prototyped using the eSensor DNA detection system. For clinical applications, the prototype assay can be readily fine-tuned by optimizing the conditions for PCR and hybridization to yield concordance levels approaching 100% (data not published).

Conclusions

The bioelectronic detection platform described here includes disposable DNA chips and laboratory-based electronic readers and is a first-generation system that ultimately will facilitate the development of relatively low cost, arrayed nucleic acid-based tests. For widespread clinical laboratory adoption, these arrays must have clinically appropriate densities compatible with the needs of molecular diagnostics laboratories, (i.e., low cost and not labor-intensive). The system described could also be developed as a foundation for devices to be used for point-of-care testing for other infectious diseases.

Declarations

Acknowledgements

Use of trade names is for identification only and does not imply endorsement by the Public Health Service or the U.S. Department of Health and Human Services. This research was supported in part by Memorandum of Understanding between the Viral Exanthems and Herpesvirus Branch in the Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention and Clinical Microsensors, A Division of Motorola and by the National Cancer Institute's Early Detection Research Network (EDRN), Interagency Agreement Y1-CN-0101-01.

Authors’ Affiliations

(1)
Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention
(2)
Motorola Life Sciences
(3)
Department of Pathology, Baylor College of Medicine
(4)
Department of Pathology, Stanford University Medical Center

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  14. Pre-publication history

    1. The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/3/12/prepub

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© Vernon et al; licensee BioMed Central Ltd. 2003

This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

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