Colleen Knoth, Melissa Henrie, Bansari Shah, Jakob Kirchner, Taihra Ul-Hasan, Derrek Mantzke, Michael Aye* Applied BioCode, Inc., Santa Fe Springs, CA 90670

Despite recent introduction of molecular multiplex pathogen detection platforms, there is limited choice of systems for clinical labs with high specimen throughput. To address this user need, Applied BioCode is developing a user friendly, high throughput automated system for multiplex molecular detection of pathogens.

The BioCode MDx 3000 platform integrates and automates PCR, post-PCR sample handling and detection steps in a 96-well format, reducing hands-on time for clinical labs. Following the extraction of nucleic acids from raw stool or Cary-Blair specimens with an automated system, DNA and RNA targets are amplified in a one-step reverse transcription-polymerase chain reaction (RT-PCR). PCR products are captured by target-specific probes coupled to unique barcoded magnetic beads (BMBs), and the presence of captured target sequence(s) is detected by streptavidin-phycoerythrin conjugate. Qualitative result for each target is determined by median fluorescence intensity (MFI) relative to the assay cutoff. Feasibility of the platform was evaluated using the BioCode gastrointestinal (GI) pathogen panel.

The BioCode GI Pathogen panel is a multiplex molecular assay based on the BMB technology for detection of gastrointestinal pathogens which include bacteria (Campylobacter, Clostridium difficile toxin A/B, Salmonella, Shigella/ enteroinvasive E. coli, enteroaggregative E. coli, enteropathogenic E. coli, enterotoxigenic E. coli, shiga toxin-producing E. coli, E. coli O157, Aeromonas, Vibrio, Yersinia enterocolitica), viruses (norovirus group I/II, adenovirus F, rotavirus A), and parasites (Cryptosporidium, Entamoeba histolytica, Giardia lamblia).

Bacteria Aeromonas spp. Campylobacter spp. (C. jejuni, C. coli) Clostridium difficile toxin A/B Escherichia coli O157 Enteroaggregative E. coli (EAEC) Enteropathogenic E. coli (EPEC) Enterotoxigenic E. coli LT/ST (ETEC) Salmonella spp. Shiga toxin producing E. coli stx1/stx2 (STEC) Shigella spp. (S. boydii, S. sonnei, S. fexneri, S. dysenteriae) /

Enteroinvasive E. coli (EIEC) Vibrio spp. and V. parahaemolyticus Yersinia enterocolitica

Parasites Cryptosporidium spp. (C. parvum, C. hominis) Entamoeba histolytica Giardia intestinalis

Viruses Adenovirus 40/41 Norovirus GI/GII Rotavirus A

Revised Abstract BioCode GI Pathogen Panel Targets

Barcoded Magnetic Bead Technology

Preliminary Limit of Detection

Robotic Arm

Heater/Mixer

Optical Stage

Reagents

Buffer & waste bottles

Tips

Thermal Cycler

Figure 1. Barcoded Magnetic Beads (BMBs) can be coupled to proteins or nucleic acids probes and used for target capture in microtiter plates. In the BioCode GI Pathogen Panel, biotinylated target DNA is captured by target-specific nucleic acid probes coupled to BMBs then labeled by SA-PE for detection.

BioCode MDx 3000

~1 hour ~0.5 hour ~3 hour

Extraction

Figure 2. Workflow for BioCode GI Pathogen Panel. 188 samples in 8 hour shift with minimal hands on time. Computer-aided drafting (CAD) image of the prototype instrument. Up to 3 different panels can be performed simultaneously in one plate.

PCR Set-Up PCR Cycling / Target Capture & SA-PE Hybridization / Optical Detection

Carryover/Precision Studies

Method Comparison

The BioCode GI Pathogen Panel and MDx 3000 are currently under development. *Corresponding author: maye@apbiocode.com

Within run carryover was not observed with the BioCode MDx 3000

Equivalent performance was observed between unpreserved stool andCary-Blair specimens

The BioCode GI Pathogen Panel did not show cross-reactivity fororganisms tested

Preliminary LOD of the panel was comparable to current IVD assays

Clinical performance with 287 stool specimens showed >90% overallagreement with the Luminex GPP assay

The BioCode MDx 3000 is an automated system for integration of PCR, post-PCR sample handling and detection steps while BioCode GI panel specifically detects several bacteria, toxins, viruses and parasites. In combination, this platform and reagents will allow users to perform multiplex molecular detection in a high throughput format, thereby simplifying the workflow, reducing hands-on time and minimizing the contamination risk.

Table 2. Results of a within run Carryover study. Potential sample-to-sample carryover or cross-contamination for the BioCode GI Pathogen Panel assayed with the BioCode MDx was examined with a DNA target (100 copies per PCR). No carryover contamination was observed. Average MFI for 48 wells was 18752; 11.7% CV. Less than 5% CV has been reported for the automated 5 µL transfer from the PCR plate to the capture plate.

Table 1. Organisms and Toxins targeted by the BioCode GI Pathogen Panel

Cross Reactivity

Table 3. Cross reactivity panel. Cross reactivity was not observed with the Microorganisms listed below.

Table 4. Preliminary Limit of Detection (LOD) for the BioCode GI Pathogen Panel.

Expanded Methods

Raw stool or Cary-Blair specimens were vortexed with beads (Precellys) in Lysis buffer prior to automated extraction with the easyMag (bioMerieux). Extracts were amplified in a one-step RT-PCR on a BioCode MDx 3000 Beta instrument. The BioCode MDx then transferred the PCR products to a hybridization plate for automated BMB target capture, streptavidin-phycoerythrin (SA-PE) conjugation and washing. Beta instruments did not have fully functional optical module at the time of this study, therefore BMB imaging and fluorescence signal detection was performed on an off-board analyzer. Qualitative results were determined by a median fluorescence intensity (MFI) signal relative to assay cutoffs.

Table 5. Comparison of BioCode GI Pathogen Panel with GPP (Luminex) results on archived raw stool specimens (n=287).

Co-infections: 52 out of the 282 specimens (18.4%) reported as positive for more than one pathogen. Invalid Samples: 5 out of 287 specimens (1.7%) gave invalid results due to failure to detect the Internal Control.

Bacteria

Parasites

Viruses

Methods

Introduction Raw Stool Vs Cary-Blair Transport Medium

Figure 3. Raw Stool Vs. Cary-Blair MFI. Equivalent signals were produced for low positive contrived samples extracted from unpreserved stool or Cary-Blair specimens.

Conclusions

1 2 3 4 5 6 7 8 9 10 11 12A 19058 38 20428 13 17065 9 21322 6 18146 9 19542 30B 31 15325 47 18493 56 19032 56 16862 9 16666 8 16707C 17256 33 16128 45 16436 40 15382 8 20723 7 18471 5D 9 18092 51 17120 64 13894 32 12888 31 17893 8 16669E 18813 15 18444 25 17950 32 18834 35 20564 5 19157 6F 9 20102 92 21665 36 20822 32 18938 10 18456 9 20532G 20432 6 19080 85 21060 51 18402 8 19923 8 21219 8H 8 23158 9 18778 7 19376 9 19354 12 21538 7 23918

Matrix Equivalency

Preliminary LOD Average MFI

8.7 x 103 CFU/mL 11783

1.0 x 103 CFU/mL 14365

3.0 x 102 CFU/mL 4896

1.6 x 104 CFU/mL 16885

1.6 x 104 CFU/mL 6774

2.5 x 103 CFU/mL 23983

1.3 x 103 CFU/mL 19361

4.0 x 103 CFU/mL 2501

4.0 x 103 CFU/mL 9383

4.0 x 104 CFU/mL 21130

1.0 x 104 CFU/mL 14887

1.0 x 103 CFU/mL 15465

5.0 x 102 CFU/mL 8354

1.3 x 103 CFU/mL 20084

1.0 x 104 CFU/mL 19947

1.4 x 101 TCID50/mL 9494

2.8 x 101 TCID50/mL 7486

3.0 x 101 TCID50/mL 18656

1.3 x 103 cysts/mL 7171

2.0 x 102 cysts/mL 36116

3.8 x 103 cysts/mL 34820

Organism

Salmonella enterica

Campylobacter jejuni

Yersinia enterocolitica

Enteroaggregative E. coli (EAEC)

Salmonella bongori

Shiga toxin producing E. coli (STEC) stx1/stx2

Enteroinvasive Escherichia coli (EIEC)

Vibrio cholerae

Vibrio parahaemolyticus

Aeromonas hydrophila

Campylobacter coli

Clostridium difficile toxin A/B

Escherichia coli O157

Enteropathogenic E. coli (EPEC)

Bacteria

Enterotoxigenic E. coli (ETEC) LT/ST

ParasitesCrytosporidium parvum

Entamoeba histolytica

Giardia lamblia (G. intestinalis )

Viruses

Adenovirus 41

Rotavirus A

Adenovirus 40

Alcaligenes faecalis Escherichia vulneris Staphylococcus aureus

Bacillus cereus Klebsiella oxytoca Staphylococcus epidermidis

Citrobacter freundii Klebsiella pneumoniae Cryptosporidium muris

Enterococcus faecalis Listeria monocytogenes Giradia muris

Enterococcus faecium Morganella morganii Coronavirus NL63

Escherichia hermannii Serratia liquefaciens Rhinovirus 1A

Pos Neg TotalPos 116 10 126Neg 11 145 156Total 127 155 282

Overall AgreementLuminex GPP Results

BioCode GI Panel

91%94%

Positive AgreementNegative Agreement

Pos Neg TotalPos 38 2 40Neg 0 242 242Total 38 244 282

Rotavirus ALuminex GPP Results

BioCode GI Panel

100%99%

Positive AgreementNegative Agreement

Pos Neg TotalPos 36 6 42Neg 5 235 240Total 41 241 282

BioCode GI Panel

Norovirus GI/GIILuminex GPP Results

88%98%

Positive AgreementNegative Agreement

Pos Neg TotalPos 38 6 44Neg 2 236 238Total 40 242 282

Clostridium difficileLuminex GPP Results

BioCode GI Panel

95%98%

Positive AgreementNegative Agreement

Pos Neg TotalPos 16 3 19Neg 2 261 263Total 18 264 282

Salmonella spp.Luminex GPP Results

BioCode GI Panel

89%99%Negative Agreement

Positive Agreement

Biocode GI Panel Luminex GPP44 400 019 186 65 N/A0 014 N/A1 00 00 00 N/A2 N/A2 20 04 N/A42 4152 N/A40 38

Entameoba histolyticaNorovirus (GI & GII)

Adenovirus (Type 40 & 41)Rotavirus A

Aeromonas spp.Vibrio spp.

Yersinia enterocoliticaGiardia lamblia

Cryptosporidium

EAECE. coli O157

EPECETECSTEC

Positive Results reported by

Clostridium difficileCampylobacter spp.

Salmonella spp.Shigella spp./ EIEC

Target Pathogens