Agpath one step real time pcr kit

Pathogens were detected among all stool samples collected from children with complete follow-up. To extract total nucleic acid, the QIAamp Fast DNA Stool Mini Kit (Qiagen) was used.17 (link) Quantitative polymerase chain reaction (qPCR) using AgPath One Step real-time PCR kit (Thermo-Fisher) was used to detect 29 enteropathogens via the TaqMan Array Card platform.1 (link) A quantification cycle threshold of 35 was the analytic limit of detection. Ten enteric pathogens that were previously identified as the top causes of diarrhoea in MAL-ED1 (link) were included in these analyses: adenovirus 40/41, astrovirus, Campylobacter jenjui/Campylobacter coli (C. jejuni/C.coli), Cryptosporidium, norovirus, rotavirus, sapovirus, Shigella, typical enteropathogenic E. coli (tEPEC) and heat stable ETEC (ST-ETEC).

Field workers collected monthly non-diarrheal stool samples without a fixative, and raw stool aliquots were stored at −80 °C before nucleic acid extraction. All laboratory testing was completed at site-specific laboratories [30 (link),31 (link)]. The QIAmp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) was used to extract total nucleic acid from stool specimens from children who had completed two years of follow-up, as previously described [32 (link)]. The efficacy of extraction and amplification was assessed using extrinsic controls, such as phocine herpesvirus (PhHV) and bacteriophage MS2. Quantitative polymerase chain reaction (PCR) with custom-designed TaqMan Array Cards was used to identify 29 enteropathogens utilizing the AgPath One Step realtime PCR kit (ThermoFisher, Waltham, MA, USA), as described elsewhere [33 (link),34 (link),35 (link)]. Shigella spp. were detected using primer sets specific for the ipaH gene [11 (link),33 (link)], and a cycle of threshold (Ct) value of less than 35 (Ct < 35) was use as the cut-off [11 (link),36 ].

The QIAmp Fast DNA Stool Mini Kit (Qiagen) was used to extract total nucleic acid from the stool specimens [8 (link)]. TaqMan Array Cards were developed to detect 29 enteropathogens via qPCR using the AgPath One Step real-time PCR kit (Thermo-Fisher), as previously detailed [8 (link)]. We specifically studied toxigenic C. difficile, and all mentions of C. difficile in the following text refer to toxigenic C. difficile. The qPCR assays targeting C. difficile TcdA [11 ] and TcdB [12 (link)] genes were validated on the TaqMan Array platform, demonstrating 100% sensitivity and 100% specificity on clinical specimens using a secondary real-time PCR as confirmation [13 (link)]. The quantification cycle for positivity by both assays was set at <35 [8 (link)]. Monthly stool samples were tested for myeloperoxidase (MPO; measured in ng/mL), neopterin (NEO; measured in nmol/L), and α-1-antitrypsin (AAT; measured in mg/g) and analyzed on the logarithmic scale. Serum α-1-acid glycoprotein (AGP; measured in mg/dL) was measured at months 7, 15, and 24. Lactulose/mannitol excretion ratios were measured in urine at 3, 6, 9, and 15 months and converted to sample-based z-scores (LMZ), as described previously [14 (link)]. Plasma zinc and retinol were measured at 7, 15, and 24 months.