fThe TaqMan Array Card (TAC) system is a 384-well singleplex real-time PCR format that has been used to detect multiple infection targets. Here , Shigella/enteroinvasive E. coli (EIEC), protozoa (Cryptosporidium, Giardia lamblia, and Entamoeba histolytica), and helminths (Ascaris lumbricoides and Trichuris trichiura), as well as two extrinsic controls to monitor extraction and amplification efficiency (the bacteriophage MS2 and phocine herpesvirus). Primers and probes were newly designed or adapted from published sources and spotted onto microfluidic cards. Fecal samples were spiked with extrinsic controls, and DNA and RNA were extracted using the QiaAmp Stool DNA minikit and the QuickGene RNA Tissue kit, respectively, and then mixed with AgPath-ID One Step real-time reverse transcription-PCR (RT-PCR) reagents and loaded into cards. PCR efficiencies were between 90% and 105%, with linearities of 0.988 to 1. The limit of detection of the assays in the TAC was within a 10-fold difference from the cognate assays performed on plates. Precision testing demonstrated a coefficient of variation of below 5% within a run and 14% between runs. Accuracy was evaluated for 109 selected clinical specimens and revealed an average sensitivity and specificity of 85% and 77%, respectively, compared with conventional methods (including microscopy, culture, and immunoassay) and 98% and 96%, respectively, compared with our laboratory-developed PCR-Luminex assays. This TAC allows fast, accurate, and quantitative detection of a broad spectrum of enteropathogens and is well suited for surveillance or clinical purposes.

Detection and quantification of enteropathogens in stool specimens is useful for diagnosing the cause of diarrhea but is technically challenging. Here we evaluate several important determinants of quantification: specimen collection, nucleic acid extraction, and extraction and amplification efficiency. First, we evaluate the molecular detection and quantification of pathogens in rectal swabs versus stool, using paired flocked rectal swabs and whole stool collected from 129 children hospitalized with diarrhea in Tanzania. Swabs generally yielded a higher quantification cycle (Cq) (average 29.7, standard deviation 3.5 vs. 25.3 ± 2.9 from stool, P<0.001) but were still able to detect 80% of pathogens with a Cq < 30 in stool. Second, a simplified total nucleic acid (TNA) extraction procedure was compared to separate DNA and RNA extractions and showed 92% (318/344) sensitivity and 98% (951/968) specificity, with no difference in Cq value for the positive results (ΔCq(DNA+RNA-TNA) = -0.01 ± 1.17, P = 0.972, N = 318). Third, we devised a quantification scheme that adjusts pathogen quantity to the specimen’s extraction and amplification efficiency, and show that this better estimates the quantity of spiked specimens than the raw target Cq. In sum, these methods for enteropathogen quantification, stool sample collection, and nucleic acid extraction will be useful for laboratories studying enteric disease.

Thirteen novel (E)-alpha-(methoxyimino)benzeneacetate derivatives, the analogues of strobilurins, which contain two pharmacophoric substructures of the methyl (E)-methoxyiminoacetate moiety and 1,3,5-substituted pyrazole ring, were stereoselectively synthesized. It was found that the coupling reaction could give stereoselectively (E:Z ca. 14:1) the key intermediate material (E)-methyl 2-(hydroxyimino)-2-o-tolyl acetate (2). An X-ray crystallographic structure determination was carried out in a representative product. The preliminary bioassays indicated that all of the compounds 1 showed potent fungicidal activity against Rhizoctonia solani, Botrytis cinereapers, Gibberella zeae, Physalospora piricola, and Bipolaris mayclis.

Salmonella infection is an important foodborne consumer health concern that can be mitigated during food processing. Bacteriophage therapy imparts many advantages over conventional chemical preservatives including pathogen specificity, natural derivation, potency, and providing a high degree of safety. The objective of this study aimed to isolate and characterize a phage that effectively control Salmonella food contamination. Out of 35 isolated phages, LPSE1 demonstrated a broad Salmonella host range, robust lytic ability, extensive pH tolerance, and prolonged thermal stability. The capacity for phage LPSE1 to control Salmonella Enteritidis-ATCC13076 in milk, sausage, and lettuce was established. Incubation of LPSE1 at 28°C in milk reduced recoverable Salmonella by approximately 1.44 log10 CFU/mL and 2.37 log10 CFU/mL at MOI of 1 and 100, respectively, as relative to the phage-excluded control. Upon administration of LPSE1 at an MOI of 1 in sausage, Salmonella count decreased 0.52 log10 at 28°C. At MOI of 100, the count decreased 0.49 log10 at 4°C. Incubation of LPSE1 on lettuce reduced recoverable Salmonella by 2.02 log10, 1.71 log10, and 1.45 log10 CFU/mL at an MOI of 1, 10, and 100, respectively, as relative to the negative control. Taken together, these findings establish LPSE1 as an effective weapon against human pathogenic Salmonella in various ready to eat foods.

Detection of an epidermal growth factor receptor (EGFR) mutation in circulating cell-free DNA (cfDNA) is a noninvasive method to collect genetic information to guide treatment of lung cancer with tyrosine-kinase inhibitors (TKIs). However, the association between cfDNA and detection of EGFR mutations in tumor tissue remains unclear. Here, a meta-analysis was performed to determine whether cfDNA could serve as a substitute for tissue specimens for the detection of EGFR mutations. The pooled sensitivity, specificity, and areas under the curve of cfDNA were 0.60, 0.94, and 0.9208 for the detection of EGFR mutations, 0.64, 0.99, and 0.9583 for detection of the exon 19 deletion, and 0.57, 0.99, and 0.9605 for the detection of the L858R mutation, respectively. Our results showed that cfDNA has a high degree of specificity to detect exon 19 deletions and L858R mutation. Due to its high specificity and noninvasive characteristics, cfDNA analysis presents a promising method to screen for mutations in NSCLC and predict patient response to EGFR-TKI treatment, dynamically assess treatment outcome, and facilitate early detection of resistance mutations.

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15The torsional properties of spider dragline silks from Nephila edulis and Nephila

The orb-weaving spider N. mellotteei has the maximum potential as a biological control agent of E. obliqua in an integrated pest management strategy. An approach of screening and evaluating main predators of insect pests through comprehensive indices was preliminarily established. © 2017 Society of Chemical Industry.