一种商业定性BAX实时PCR检测方法的适应性研究，用于定量全禽胴体冲洗液中的 spp.。

Adaptation of a Commercial Qualitative BAX Real-Time PCR Assay to Quantify spp. in Whole Bird Carcass Rinses.

作者信息

Bodie Aaron R, Dittoe Dana K, Applegate Savannah F, Stephens Tyler P, Ricke Steven C

机构信息

Meat Science and Animal Biologics Discovery Program, Department of Animal and Dairy Sciences, University of Wisconsin, Madison, WI 53706, USA.

Department of Animal Science, University of Wyoming, Laramie, WY 82071, USA.

出版信息

Foods. 2023 Dec 22;13(1):56. doi: 10.3390/foods13010056.

DOI:10.3390/foods13010056

PMID:38201085

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10778266/

Abstract

Poultry is the primary reservoir of , a leading cause of gastroenteritis in the United States. Currently, the selective plating methodology using selective agars, Campy Cefex and Modified Charcoal Cefoperazone Deoxycholate agar, is preferentially used for the quantification of spp. among poultry products. Due to the specific nature of , this methodology is not sensitive, which can lead to skewed detection and quantification results. Therefore, detection and quantification methods are urgently needed. The objective was to develop a shortened enrichment-based quantification method for (CampyQuant™) in post-chill poultry rinsates using the BAX System Real-Time PCR assay for . The specificity and sensitivity for the detection of , , and in pure culture were determined. The BAX System Real-Time PCR assay consistently detected and identified each species 100% of the time with an enumeration range of 4.00 to 9.00 Log CFU/mL. Enrichment time parameters for low-level concentrations (0.00, 1.00, and 2.00 Log CFU/mL) of using the BAX System Real-Time PCR assay were elucidated. It was determined that an enrichment time of 20 h was needed to detect at least 1.00 Log CFU/mL of spp. Using the BAX System Real-Time PCR assay for . As a result, time of detection, detection limits, and enrichment parameters were used to develop the CampyQuant™ linear standard curve using the detected samples from the BAX System Real-Time PCR assay to quantify the levels in post-chill poultry rinsates. A linear fit equation was generated for each species using the cycle threshold from the BAX System Real-Time PCR assay to estimate a pre-enrichment of 1.00 to 4.00 Log CFU/mL of rinsates detected. The statistical analyses of each equation yielded an R of 0.93, 0.76, and 0.94 with a Log RMSE of 0.64, 1.09, and 0.81 from , , and , respectively. The study suggests that the BAX System Real-Time PCR assay for is a more rapid, accurate, and efficient alternative method for enumeration.

摘要

家禽是在美国导致肠胃炎的主要病因——弯曲杆菌的主要宿主。目前，使用选择性琼脂、弯曲杆菌头孢菌素（Campy Cefex）和改良的含头孢哌酮脱氧胆酸盐的木炭琼脂的选择性平板接种方法，优先用于家禽产品中弯曲杆菌属细菌的定量分析。由于弯曲杆菌的特殊性质，这种方法并不灵敏，可能会导致检测和定量结果出现偏差。因此，迫切需要弯曲杆菌的检测和定量方法。目的是开发一种基于缩短增菌的定量方法（CampyQuant™），用于检测冷却后家禽冲洗液中的弯曲杆菌，该方法使用BAX系统实时荧光定量PCR检测弯曲杆菌。测定了该方法对纯培养物中弯曲杆菌、空肠弯曲杆菌和结肠弯曲杆菌检测的特异性和灵敏度。BAX系统实时荧光定量PCR检测方法始终能100%地检测和鉴定出每种菌株，其计数范围为4.00至9.00 Log CFU/mL。阐明了使用BAX系统实时荧光定量PCR检测低浓度（0.00、1.00和2.00 Log CFU/mL）弯曲杆菌的增菌时间参数。确定需要20小时的增菌时间才能检测到至少1.00 Log CFU/mL的弯曲杆菌属细菌。使用BAX系统实时荧光定量PCR检测弯曲杆菌。结果，利用检测时间、检测限和增菌参数，使用BAX系统实时荧光定量PCR检测的样本建立了CampyQuant™线性标准曲线，以定量冷却后家禽冲洗液中的细菌水平。使用BAX系统实时荧光定量PCR的循环阈值，为每种弯曲杆菌菌株生成线性拟合方程，以估计检测到的冲洗液中1.00至4.00 Log CFU/mL的预增菌量。对每个方程的统计分析得出，弯曲杆菌、空肠弯曲杆菌和结肠弯曲杆菌的R值分别为0.93、0.76和0.94，对数均方根误差（Log RMSE）分别为0.64、1.09和0.81。该研究表明，用于弯曲杆菌检测的BAX系统实时荧光定量PCR检测方法是一种更快速、准确和高效的弯曲杆菌计数替代方法。