This document describes the development of multiplex PCR panels to rapidly detect all major gastrointestinal pathogens from a single patient sample in less than 3 hours. A universal extraction method was developed that can simultaneously extract nucleic acids from bacteria, viruses, and parasites from fecal samples. PCR assays were developed that target the major gastrointestinal pathogens and include controls. Clinical testing demonstrated excellent agreement with conventional diagnostic methods and improved performance over some existing molecular assays. The developed assays provide a simplified screening system for comprehensive gastrointestinal pathogen detection directly from patient samples.

1. Multiplex PCR detection of all major gastrointestinal pathogens
employing a novel universal extraction method
J.R. Melki*, S.P. Siah, K. Kaur, J. Nair, N. Coulston, D.S. Millar (Sydney, AU)
Figure 3. Amplification plots obtained using
Abstract Materials & Methods Results the EasyScreen™ Viral assay.
Gastrointestinal disease (GI) is a major A universal sample processing method was All assays were linear from 106-10 copies
cause of morbidity and mortality world- devised that lysed and simultaneously and no cross-reactivity was observed
wide. GI can be caused by a wide range of converted the nucleic acids of bacteria, between individual primers and a larger
Sapovirus Rotavirus
infectious agents including viral, bacterial viruses and protozoan parasites from the number of bacterial and fungal non-target
and protozoa. Human viral gastroenteritis primary patient sample. The sample buffer species. Over 500 clinical samples have
can be caused Noroviruses, Rotaviruses, protects the labile RNA species from the been assessed and compared to
Adenoviruses, Astroviruses and Sapovirus. harsh conditions required for complete lysis conventional techniques such as culture,
Of these Norovirus is the most commonly of tough organisms such as Giardia. The EIA and microscopy with excellent Norovirus gpII Adenovirus
isolated agent as the cause of acute viral procedure comprises a single tube method concordance. The method developed here
gastroenteritis (1). According to the CDC during which the faecal sample is incubated is therefore suitable for the rapid and
43% of bacterial GI infections are caused in extraction buffer for 15 minutes. Samples sensitive screening of primary patient
by Salmonella, followed by Campylobacter were then purified using a column-based material for a wide range of common GI
(33%), Shigella (17%), Escherichia coli method or automated platforms such as pathogens. Astrovirus Norovirus gpI
(4.1%) and Yersinia (0.9%). Another cause Roche, Qiagen and ThermoFisher. PCR Table 4. Results obtained using the
of bacterial GI are hypervirulent strains of can then be carried out on most real-time Figure 2. Amplification plots obtained using EasyScreen™ Viral assay.
Clostridium difficile particularly PCR instruments including those from Roche, the EasyScreen™ Parasite assay.
™
™
ribotype 027 (2). Among parasites Giardia Qiagen, Cepheid, ABI, Biorad and
intestinalis, Cryptosporidium spp and Stratagene. All reagents required from
Entamoeba histolytica are considered the sample to result are included simplifying the
most common and important causes of method for the end user.
diarrhea (3) although other species such as Giardia Entamoeba
*The Rotavirus EIA cross reacts with the vaccine strain
Dientamoeba fragilis and Blastocystis Table 5. Results obtained using the
Figure 1. Amplification plots obtained using the
hominis have also been implicated in GI EasyScreen™ Bacterial assay.
C. difficile EasyScreen™ Detection kit.
disease. Thus the diagnosis of GI can be
challenging and involve specialists in
microbiology, virology and parasitology. Cryptosporidium Blastocystis hominis
Dientamoeba fragilis Extraction Control
Objective Table 3. Results obtained using the Discussion
EasyScreen™ Parasite assay.
In order to simplify the detection of Table 2. Three independent cllinical studies The assays developed here may be
using the EasyScreen™ C. diffiicle Detection
causative agents of GI we have developed used as a complete screening system
assay.
rapid real time multiplex PCR (mPCR) for the diagnosis of all major GI
panels for all major GI pathogens (see pathogens from primary clinical samples.
Table 1). All assays share a universal The assays are simple and employ
sample processing method and incorporate universal sample preparation conditions
our previously described 3base™ Using the EasyScreen™ GI panels we thereby streamlining the process of
technology (4). Furthermore, conventional consistently achieved better results than pathogen detection from faecal material.
GI diagnosis can in some instances take up conventional techniques such as culture, All assays have incorporated controls for
to 5 days (5) to provide a definitive result. microscopy and EIA. The EasyScreen™ sample processing and inhibition to
To reduce this time we aimed to produce C.difficile Detection kit demonstrated 100% ensure assay robustness and reliability.
assays with sample to result turnaround concordance when tested against the The assays can be run on virtually all
time in as little as 3 hours. “Gold-Standard” of Culture Toxin (see purification and real-time instruments
Table 2). In addition the method also found in major hospital and pathology
showed improved performance when laboratories. Sample to results time is
tested against two independent molecular less than 3 hours, allowing for rapid
assays. The EasyScreen™ Parasite assay diagnosis facilitating optimal patient
also demonstrate improved detection when management.
compared to Microscopy and EIA (see
*Not toxigenic culture Table 3). References
Table 1. Targets detected by the EasyScreen™ GI Panels. 1. Estes MK, Prasad BV, Atmar RI. 2006. Noroviruses
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WN, Goorhuis B, Kuijper EJ, Wilcox MH. 2010. The
changing epidemiology of Clostridium difficile infections.
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4. Baleriola C, Millar D, Melki J, Coulston N, Altman P,
Rismanto N and Rawlinson W. 2008. Comparison of a
novel HPV test with the Hybrid Capture II (hcII) and a
reference PCR method shows high specificity and positive
predictive value for 13 high-risk human papillomavirus
E
EC001 infections. J. Clin. Virol. 42:22-6.
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*Coming soon Mandrekar J and Patel R. 2010. Three-hour molecular
detection of Campylobacter, Salmonella, Yersinia and
Shigella species in feces with accuracy as high as that of
culture. J. Clin. Micro. 48: 2929-33