Development, optimization, and validation of a \u3ci\u3eClassical swine fever virus\u3c/i\u3e real-time reverse transcription polymerase chain reaction assay

Classical swine fever (CSF) is an economically devastating disease of pigs. Instrumental to the control of CSF is a well-characterized assay that can deliver a rapid, accurate diagnosis prior to the onset of clinical signs. A real-time fluorogenicprobe hydrolysis (TaqMan) reverse transcription polymerase chain reaction (RT-PCR) for CSF was developed by the United States Department of Agriculture (USDA) at the Plum Island Animal Disease Center (CSF PIADC assay) and evaluated for analytical and diagnostic sensitivity and specificity. A well-characterized panel including Classical swine fever virus (CSFV), Bovine viral diarrhea virus (BVDV), and Border disease virus (BDV) isolates was utilized in initial feasibility and optimization studies. The assay was initially designed and validated for use on the ABI 7900HT using the Qiagen QuantiTect® Probe RT-PCR chemistry. However, demonstrating equivalency with multiple one-step RT-PCR chemistries and PCR platforms increased the versatility of the assay. Limit of detection experiments indicated that the Qiagen QuantiTect® Multiplex (NoROX) and the Invitrogen SuperScript® III RT-PCR kits were consistently the most sensitive one-step chemistries for use with the CSF PIADC primer/probe set. Analytical sensitivity of the CSF PIADC assay ranged from \u3c1–2.95 log10 TCID50 /ml on both the ABI 7900HT and ABI 7500 platforms. The CSF PIADC assay had 100% diagnostic sensitivity and specificity when tested on a panel of 152 clinical samples from the Dominican Republic and Colombia. The ability to perform this newly developed assay in 96-well formats provides an increased level of versatility for use in CSF surveillance programs

Suggested guidelines for validation of real-time PCR assays in veterinary diagnostic laboratories

This consensus document presents the suggested guidelines developed by the Laboratory Technology Committee (LTC) of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) for development, validation, and modification (methods comparability) of real-time PCR (rtPCR) assays. These suggested guidelines are presented with reference to the World Organisation for Animal Health (OIE) guidelines for validation of nucleic acid detection assays used in veterinary diagnostic laboratories. Additionally, our proposed practices are compared to the guidelines from the Foods Program Regulatory Subdivision of the U.S. Food and Drug Administration (FDA) and from the American Society for Veterinary Clinical Pathology (ASVCP). The LTC suggestions are closely aligned with those from the OIE and comply with version 2021-01 of the AAVLD Requirements for an Accredited Veterinary Medical Diagnostic Laboratory, although some LTC recommendations are more stringent and extend beyond the AAVLD requirements. LTC suggested guidelines are substantially different than the guidelines recently published by the U.S. FDA for validation and modification of regulated tests used for detection of pathogens in pet food and animal-derived products, such as dairy. Veterinary diagnostic laboratories that perform assays from the FDA Bacteriological Analytical Method (BAM) manual must be aware of the different standard

Body-composition changes in the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE)-2 study: A 2-y randomized controlled trial of calorie restriction in nonobese humans

Background: Calorie restriction (CR) retards aging and increases longevity in many animal models. However, it is unclear whether CR can be implemented in humans without adverse effects on body composition.Objective: We evaluated the effect of a 2-y CR regimen on body composition including the influence of sex and body mass index (BMI; in kg/m2) among participants enrolled in CALERIE-2 (Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy), a multicenter, randomized controlled trial.Design: Participants were 218 nonobese (BMI: 21.9-28.0) adults aged 21-51 y who were randomly assigned to 25% CR (CR, n = 143) or ad libitum control (AL, n = 75) in a 2:1 ratio. Measures at baseline and 12 and 24 mo included body weight, waist circumference, fat mass (FM), fat-free mass (FFM), and appendicular mass by dual-energy X-ray absorptiometry; activity-related energy expenditure (AREE) by doubly labeled water; and dietary protein intake by self-report. Values are expressed as means ± SDs.Results: The CR group achieved 11.9% ± 0.7% CR over 2-y and had significant decreases in weight (-7.6 ± 0.3 compared with 0.4 ± 0.5 kg), waist circumference (-6.2 ± 0.4 compared with 0.9 ± 0.5 cm), FM (-5.4 ± 0.3 compared with 0.5 ± 0.4 kg), and FFM (-2.0 ± 0.2 compared with -0.0 ± 0.2 kg) at 24 mo relative to the AL group (all between-group P < 0.001). Moreover, FFM as a percentage of body weight at 24 mo was higher, and percentage of FM was lower in the CR group than in the AL. AREE, but not protein intake, predicted preservation of FFM during CR (P < 0.01). Men in the CR group lost significantly more trunk fat (P = 0.03) and FFM expressed as a percentage of weight loss (P < 0.001) than women in the CR group.Conclusions: Two years of CR had broadly favorable effects on both whole-body and regional adiposity that could facilitate health span in humans. The decrements in FFM were commensurate with the reduced body mass; although men in the CR group lost more FFM than the women did, the percentage of FFM in the men in the CR group was higher than at baseline. CALERIE was registered at clinicaltrials.gov as NCT00427193