Department of Food, Nutrition and Packaging Sciences, Clemson University, SC 29634, USA.
Department of Food, Nutrition and Packaging Sciences, Clemson University, SC 29634, USA.
Poult Sci. 2023 Feb;102(2):102369. doi: 10.1016/j.psj.2022.102369. Epub 2022 Dec 6.
Polymerase chain reaction (PCR) method was coupled with a DNA extraction to enumerate Campylobacter spp. from poultry gastrointestinal tract samples. Three experiments were conducted that included: 1) Development of a DNA standard curve related to bacterial DNA primers; 2) Design of a cell/genomic DNA extraction protocol to isolate Campylobacter spp. DNA from complex samples such as poultry feces; and 3) Comparison of PCR quantification to standard plate count methodology. The standard curve using primers for Campylobacter spp. was created for DNA extracted from environmental isolates with a linear range (R > 0.95) and with a high specificity for C. coli and C. jejuni recovered from poultry, swine and laboratory isolates. A 2-step extraction process of bacterial DNA from poultry feces was developed in which the cells were first concentrated using a gradient-centrifugation step followed by comparison of 4 DNA extraction methods. Two commercial DNA extraction methods (Zymo Research Quick DNA, and Invitrogen magnetic separation), a traditional phenol-chloroform DNA extraction method using proteinase K to inactivate DNAses, and an in-house isolation method for DNA extraction based on chaotropic salts were used. The middle gradient layer recovered 89% to 98% of the bacteria cells from the sample, with recovery dependent upon the Campylobacter genus. The 4 DNA extractions methods recovered 112 to 302 ug/nL of DNA. Finally, the qPCR and standard plate methods were highly correlated for enumerating Campylobacter spp. in the 2.0 to 8.0-log CFU range. Analyses of the results from this study demonstrate that the combination of the standard curve for Campylobacter spp. DNA primers, the gradient cell concentration method and DNA extraction techniques with qPCR can be used to enumerate Campylobacter spp. from poultry samples with findings similar those of traditional plate count methodology.
聚合酶链反应 (PCR) 方法与 DNA 提取相结合，用于从家禽胃肠道样本中计数弯曲菌属。进行了三项实验，包括：1）建立与细菌 DNA 引物相关的 DNA 标准曲线；2）设计一种细胞/基因组 DNA 提取方案，从家禽粪便等复杂样本中分离弯曲菌属 DNA；3）比较 PCR 定量与标准平板计数方法。使用弯曲菌属引物的标准曲线是为从环境分离株中提取的 DNA 创建的，具有线性范围（R>0.95），并且对从家禽、猪和实验室分离株中回收的 C. coli 和 C. jejuni 具有高特异性。开发了一种从家禽粪便中提取细菌 DNA 的 2 步提取过程，其中首先使用梯度离心步骤浓缩细胞，然后比较 4 种 DNA 提取方法。使用了 2 种商用 DNA 提取方法（Zymo Research Quick DNA 和 Invitrogen 磁分离）、一种使用蛋白酶 K 灭活 DNAses 的传统酚氯仿 DNA 提取方法，以及一种基于嗜热盐的 DNA 提取的内部分离方法。中间梯度层从样品中回收了 89%至 98%的细菌细胞，回收取决于弯曲菌属。4 种 DNA 提取方法回收了 112 至 302 ug/nL 的 DNA。最后，qPCR 和标准平板方法在计数 2.0 至 8.0-log CFU 范围内的弯曲菌属方面高度相关。本研究结果分析表明，弯曲菌属 DNA 引物的标准曲线、梯度细胞浓缩方法和与 qPCR 结合的 DNA 提取技术可用于从家禽样品中计数弯曲菌属，其结果与传统平板计数方法相似。
