DiagCORE® Gastrointestinal Panel: a sensitive real time RT-qPCR -based approach to syndromic testing

Background: DiagCORE® Gastrointestinal Panel (DC-GI) is a cartridge-based, simple, easy to use, and fast syndromic testing assay that tests for the presence or absence of 24 Gastrointestinal pathogens known to cause gastrointestinal syndromes. The assay includes 6 viruses, 14 bacteria and 4parasites, and uses a 200 ml stool sample resuspended in Cary Blair medium. The DC-GI has an integrated pathogen lysis and nucleic acid purification process followed by multiplex hydrolysis probe- based real-time RT-PCR. DC-GI has been optimized to detect difficult-to-lyse organisms such as parasitical oocysts or gram positive bacterial targets, as well as gram-negative bacterial and viral targets. In addition, user can check the amplification curves of real time RT-PCR, being able to assess relative load of the targets detected. The efficacy of syndromic testing depends on the analytical sensitivity and specificity characteristics of the assay and the current study aimed to determine analytical performance of the DC-GE.

Materials/methods: All organisms were tested in several concentrations using 200 DC-GI cartridges using 6 pools of up to 6 organisms (combined samples) per pool. With this approach up to 34 results were generated for each organism. Commercially available pre-quantified stocks were used for 19 pathogens. Additional testing for targets commercially not available (5) was done using clinical samples.

Each pool was serially diluted and spiked into 5 unique clinical negative stool samples resuspended in Cary Blair (matrix) to determine whether multiple co-infecting pathogens can be detected within a single sample, as well as to characterize the effects of inhibition and different matrices

Results: DC-GI shows a sensitivity ranging from 102 to 104 cells, or viral particles per milliliter of the pathogens tested. All samples completed testing with no loss due to inhibition or fluidic failure.

Conclusions: The sensitivity of the DC-GI reaches as low as 100 pathogens/mL for several of the pathogens tested. These results highlight that a high level of sensitivity can been achieved in a sample matrix that is rich with commensal organisms, human cells resulting in high levels of ´non-target´nucleic acids. In addition the DC-GI showed excellent reliability without the need of sample preparation steps.

 

Josep Pareja, PhD (jpareja@stat-dx.com), Marta Bas, MSc (mbas@stat-dx.com), Pau Boher, PhD (pboher@stat-dx.com), Neus Guillen, MSc (nguillen@stat-dx.com), Marta López-Fontanals. PhD* (mlopez@stat-dx.com)

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