multiplex molecular gastrointestinal pathogen detection with an...
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Multiplex Molecular Gastrointestinal Pathogen Detection with an Automated, High Throughput System Jakob Kirchner, Colleen Knoth, Melissa Henrie, Bansari Shah, Taihra Ul-Hasan, Derrek Mantzke, Marla Saclolo, Michael Aye*
Applied BioCode, Inc., Santa Fe Springs, CA 90670, USA
Gastroenteritis is the second most common illness after the common cold. Globally, diarrhea accounts for approximately 2 million annual deaths in children under 5 years old, or 19% of total child deaths.1 High-throughput multiplex assays can aid in rapid identification of pathogens that can cause outbreaks of diarrhea and for infection control in healthcare settings. Using the proprietary barcoded magnetic bead (BMB) technology, Applied BioCode has developed a molecular diagnostic assay for detection of gastrointestinal pathogens. In parallel, we have developed an automated high-throughput system with a 96-well format.
Reference: 1. Boschi-Pinto C, Velebit L, Shibuya K. Estimating child mortality due to diarrhoea in developing countries. Bull World Health Organ. 2008; 86(9):710–7.
Abstract BioCode GI Pathogen Panel Targets
Barcoded Magnetic Bead Technology
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.
Figure 2. Workflow for BioCode GI Pathogen Panel. 188 samples in 8 hour shift with minimal hands-on time. Up to 3 different assays can be performed simultaneously in one plate.
Instrument Capabilities
The BioCode GI Pathogen Panel and BioCode MDx 3000 platform are currently under development. *Corresponding author: [email protected]
Table 1. Organisms and Toxins targeted by the BioCode GI Pathogen Panel
♦ Campylobacter spp.♦ Clostridium difficile toxin A/B♦ E. coli O157♦ Enteroaggregative E. coli (EAEC)♦ Enteropathogenic E. coli (EPEC)♦ Enterotoxigenic E. coli (ETEC)♦ Salmonella spp.♦ Shiga-like toxin-producing E. coli (STEC)♦ Shigella/Enteroinvasive E. coli (EIEC)♦ Vibrio parahaemolyticus♦ Vibrio spp.♦ Yersinia enterocolitica
Expanded Methods
Methods
Introduction
Bacteria Parasites
♦ Cryptosporidium spp.♦ Entamoeba histolytica♦ Giardia lamblia
♦ Adenovirus 40/41♦ Norovirus GI/GII♦ Rotavirus A
Viruses
~0.5 hours
Extraction PCR Set-Up
PCR Cycling / Target Capture and SA-PE Hybridization /
Optical Detection
~3.5 hours ~1 hour
The BioCode MDx 3000 platform integrates and automates PCR, post-PCR sample handling and detection steps in a 96-well format. Following extraction of nucleic acids with an automated system, DNA and RNA targets were amplified by RT-PCR. PCR products were captured by target-specific probes coupled to BMBs, and the presence of target sequence(s) was detected by a fluorescent conjugate. Qualitative results were determined by a median fluorescence index (MFI) value relative to assay cutoff.
The BioCode GI Pathogen Panel is an 18-plex molecular assay for detection of gastrointestinal pathogens which include bacteria (Campylobacter, C. 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, Vibrio, Yersinia enterocolitica), viruses (norovirus group I/II, adenovirus F, rotavirus A), and parasites (Cryptosporidium, Entamoeba histolytica, Giardia lamblia).
BioCode MDx 3000
Method Comparison Carryover/ Precision Study
Cross Reactivity
Preliminary Limit of Detection (LoD)
Conclusions
Table 4. Organisms tested for cross reactivity. No cross reactivity was observed.
Bacteria
Aeromonas species E. coli (non- pathogenic) Plesiomonas shigelloides
Acinetobacter baumannii Escherichia hermannii Proteus penneri
Acinetobacter lwoffii Escherichia vulneris Proteus vulgaris
Alcaligenes faecalis Fusobacterium varium Providencia species
Bacillus cereus Gemella morbillorum Pseudomonas aeruginosa
Bifidobacterium breve Klebsiella oxytoca Saccharomyces boulardii
Candida albicans Klebsiella pneumoniae Serratia liquefaciens
Cedecea davisae Lactobacillus acidophilus Serratia marcescens
Chlamydia trachomatis Lactobacillus reuteri Shewanella algae
Citrobacter freundii Lactococcus lactis Staphylococcus aureus
C. difficile (non-toxigenic) Leminorella grimontii Staphylococcus epidermidis
Clostridium sp. (non-toxigenic) Listeria monocytogenes Streptococcus salivarius
Clostridium tetani Morganella morganii Vibrio flauvialis
Viruses Parasites
Adenovirus Types: 3,4,7a,8,14,37
Cytomegalovirus (CMV) Cryptosporidium meleagridis
Coronavirus 229E Enterovirus 68 Giardia muris
Coronavirus NL63 Rhinovirus 1A Blastocyctis hominis
Coxsackievirus B3 Cryptosporidium muris Pentatrichomonas hominis
The BioCode MDx 3000 instrument integrates PCR, post-PCR handling and optical detection. The BioCode GI Pathogen Panel specifically detects several bacteria, toxins, viruses and parasites. Combined, the instrument and reagents, allow users to perform multiplex molecular detection in a high-throughput, automated, format simplifying workflow, reducing hands-on time and minimizing contamination risk.
♦ No cross-contamination observed for assays run on the BioCode MDx 3000 platform.
♦ Preliminary LOD of the BioCode GI Panel is comparable to current IVD assays.
♦ The BioCode GI Panel showed no cross-reactivity with gut flora and non-gutpathogens. This panel detects Adeno 40/41 and doesn’t cross react with otherAdenovirus types.
♦ Clinical performance with 287 stool specimens showed >90% overall agreementwith the Luminex GPP assay.
Target Pathogens
Positive Results reported by
BioCode GI Panel Luminex GPP
Clostridium difficile 44 40
Campylobacter spp. 0 0
Salmonella spp. 19 18
Shigella spp./ EIEC 6 6
EAEC 5 N/A
E. coli O157 0 0
EPEC 14 N/A
ETEC 1 0
STEC 0 0
Vibrio spp. 0 N/A
Yersinia enterocolitica 2 N/A
Giardia lamblia 2 2
Cryptosporidium 0 0
Entameoba histolytica 4 N/A
Norovirus (GI & GII) 42 41
Adenovirus (Type 40 & 41) 52 N/A
Rotavirus A 40 38
Overall Agreement Luminex GPP Results
Pos Neg Total
BioCode GI Pathogen Panel
Pos 116 10 126
Neg 11 145 156
Total 127 155 282
Positive Agreement 91%
Negative Agreement 94%
H
G
F
E
D
C
B
A
0
50000
1 2 3 4 5 6 7 8 9 10 11 12
MFI
Potential for sample-to-sample carryover or cross-contamination for the BioCode MDx 3000 (from PCR to optical detection) was examined with 100 copies of genomic DNA per PCR in a checker board pattern.
♦ Average MFI for 48 wells with genomic DNA was 18752, and 11.7% CV.
0
10000
20000
30000
40000
50000
60000
0 10000 20000 30000 40000 50000 60000
Exp
ect
ed
MFI
Observed MFI
Raw stool specimens were vortexed with beads (Precellys) in easyMag Lysis buffer prior to automated extraction with the easyMag (biomerieux). Extracts were amplified in a one-step RT-PCR on a BioCode MDx 3000. The BioCode MDx 3000 then transferred the PCR products to a hybridization plate for automated target capture, streptavidin-phycoerythrin (SA-PE) conjugation and washing. Optical detection for some experiments, including for method comparison, were performed on an off-board plate reader. Qualitative results were determined by a median fluorescence intensity (MFI) signal relative to assay cutoffs.
BioCode MDx 3000 Robotics/ Liquid Handling:
♦ Liquid dispensing: 10% CV for 5 µL volume
♦ BMB distribution: 11 %CV across 96 wells for BMB mixed in a vial anddispensed by robot.
BioCode MDx 3000 Optics:
♦ BMB decoding accuracy: 100% for different BMBs tested.
♦ BMB decoding rate: Average decoding rate is >83.4 for BMBs tested.
♦ MFI is reproducible and linear between 700 to 50,000 for duplicate runson 2 instruments over 4 days (See graph below).
Figure 3. Linearity of BioCode MDx 3000 optical detection. Observed vs. Expected MFI data from two different beta instruments.
Table 3. Overall Clinical Agreement: BioCode GI Pathogen Panel vs. Luminex GPP
Figure 4. MFI values from a carryover contamination study. No carryover contamination was observed across multiple experiments.
Organism Preliminary LOD
Bacteria
Campylobacter coli 1.0 x 103 CFU/mL
Campylobacter jejuni 7.2 x 103 CFU/mL
Clostridium difficile toxin A/B 8.0 x 103 CFU/mL
Escherichia coli O157 1.6 x 104 CFU/mL
Enteroaggregative E. coli (EAEC) 3.0 x 104 CFU/mL
Enteroinvasive Escherichia coli (EIEC) 1.3 x 103 CFU/mL
Enteropathogenic E. coli (EPEC) 4.0 x 103 CFU/mL
Enterotoxigenic E. coli (ETEC) LT/ST 7.5 x 103 CFU/mL
Salmonella bongori 4.0 x 104 CFU/mL
Salmonella enterica 1.3 x 104 CFU/mL
Shiga toxin producing E. coli (STEC) stx1/stx2
1.5 x 104 CFU/mL
Vibrio cholerae 5.0 x 102 CFU/mL
Vibrio parahaemolyticus 6.5 x 102 CFU/mL
Yersinia enterocolitica 5.0 x 103 CFU/mL
Viruses
Adenovirus 40 1.5 x 101 TCID50/mL
Adenovirus 41 2.5 x 102 TCID50/mL
Norovirus GI 4.1 x 103 TCID50/mL
Norovirus GII 3.8 x 102 TCID50/mL
Rotavirus A 1.5 x 101 TCID50/mL
Parasites
Cryptosporidium parvum 6.3 x 103 cysts/mL
Entamoeba histolytica 1.0 x 102 cysts/mL
Giardia lamblia (G. intestinalis) 1.9 x 103 cysts/mL
Table 2. Method Comparison: BioCode GI Pathogen Panel vs. Luminex GPP
Bacteria tested at 106 CFU/mL, viruses at 105 TCID50/mL, parasites at 105 cells or cysts /mL
Table 5. Preliminary LoD of GI Pathogen Panel tested on BioCode MDx 3000
52 of 282 specimens (18.4%) reported as co-infections. 5 of 287 specimens (1.7%) gave an invalid result due to failure of internal control.
Barcoded Magnetic Bead