Custom-developed TaqMan Array Cards (TAC) were utilized for detection of infections in stool. Briefly, RNA and DNA were combined and tested using the methodology, master mix, and cycling conditions previously described on a Viia7 platform (Life Technologies, South San Francisco, CA) [13] (link). Validation of the platform has been described [14] (link). We tested for alphabetically, the following infections using those previously described assays with additions as specified: adenovirus 40/41 [20] (link), Aeromonas spp. [21] (link), Ancylostoma duodenale[22] (link), Ascaris lumbricoides, astrovirus, Bacteroides fragilis[23] (link), Campylobacter spp. [24] (link), Clostridium difficile, Cryptosporidium hominus/parvum[25] (link), Cyclospora cayetanensis[26] (link), Cystoisospora belli[27] (link), Encephalitozoon intestinalis[28] (link), Entamoeba histolytica, Enterocytozoon bieneusi[28] (link), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC)/Shigella, enteropathogenic E. coli (EPEC), enterotoxigenic E.coli (ETEC), pan-enterovirus [29] (link), Giardia lamblia, Helicobacter pylori, Mycobacterium tuberculosis[30] (link), Necator americanus[22] (link), norovirus GI [31] (link), norovirus GII, rotavirus, Salmonella spp., sapovirus, Shiga-toxin producing E. coli (STEC), Strongyloides stercoralis[32] (link)), Trichuris trichiura and Vibrio cholerae[21] (link). Virulence genes were used to define the E. coli pathotypes as follows: aaiC and/or aatA for EAEC, ipaH for EIEC/Shigella, ST and/or LT for ETEC, eae with or without bfpA for EPEC, and stx1 and/or stx2 for STEC. Amplification after threshold cycle (Ct) above 35 was considered negative. In addition, all available 10 week stool from children who received RV per protocol and were followed until at least one year of age were tested by the cognate pan-EV RT-qPCR on plates. Briefly, 0.8 μl Ag-Path One-Step RT enzyme (Life Technologies, South San Francisco, CA), 10 μl Ag-Path 2× buffer, 7.2 μl nuclease free water, 1 μl of enterovirus assay (primer and probe mix) and 1 μl of RNA was tested in 20 μl reaction and run on a CFX cycler (Bio-Rad, Ventura, CA) with cycling conditions: 45 °C for 20 min, 95 °C for 10 min, and 45 cycles of 95 °C for 15 s and 60 °C for 1 min. To further describe EV infections in these stools, we utilized a multiplex RT-qPCR assay to identify Sabin strain polioviruses [33] (link).
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