Bussalleu E, Althouse G C
Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology, University of Girona, 17071 Girona, Catalonia, Spain.
Department of Clinical Studies, New Bolton Center, University of Pennsylvania, 382 West Street Road, Kennett Square, PA 19348-1692, USA.
J Microbiol Methods. 2018 Aug;151:106-110. doi: 10.1016/j.mimet.2018.06.012. Epub 2018 Jun 28.
Serratia marcescens is a bacterial contaminant that can be spermicidal when present in extended boar semen that is typically stored prior to breeding use at 15 to 18 °C for several days. This particular contaminant appears to originate from carrier boars, where it resides in the preputial cavity, but has also been shown to then easily contaminate the semen-processing laboratory. Screening for carrier boars to date has been performed through detection of S.marcescens in ejaculates using traditional agar plate culture techniques. These agar growth techniques are labor and time consuming due to the need for sample °titration and temporal growth followed by isolation, leading to delays in identification. The aim of this study was to develop a rapid, sensitive traditional PCR technique that can detect the presence of S.marcescens in extended boar semen. Primers for the detection of S. marcescens 16S rRNA were designed and specificity tested. After PCR optimization, assay sensitivity was evaluated using extended boar semen that was inoculated with various physiological ratios of spermatozoa: S.marcescens (100:1, 50:1, 20:1, 10:1, 8:1, 6:1, 4:1, 2:1. 1:1 and 1:10). Samples, held at 16 °C, were tested every 24 h over a 96 h period, with bacterial DNA extraction performed at each time point using a commercial kit. As a final step, the developed technique was used to screen random samples of extended boar semen for S. marcescens contamination. Results showed that this PCR technique had a sensitivity (90%) and specificity (100%) at detecting S.marcescens in the different inoculated ratios as well as in random, naturally contaminated samples of extended boar semen. In conclusion, this study reports a traditional PCR technique that is effective at rapidly and accurately detecting the presence of S.marcescens in boar extended semen.
粘质沙雷氏菌是一种细菌污染物，当它存在于公猪的稀释精液中时具有杀精作用。这种精液通常在配种前于15至18°C下储存数天。这种特定的污染物似乎源自携带病菌的公猪，病菌存在于包皮腔内，但也已表明它很容易污染精液处理实验室。迄今为止，通过使用传统琼脂平板培养技术检测射精中的粘质沙雷氏菌来对公猪携带者进行筛查。由于需要进行样品滴定和随时间观察生长情况，然后进行分离，这些琼脂生长技术既费力又耗时，导致鉴定延迟。本研究的目的是开发一种快速、灵敏的传统PCR技术，该技术可以检测公猪稀释精液中粘质沙雷氏菌的存在。设计了用于检测粘质沙雷氏菌16S rRNA的引物并进行了特异性测试。经过PCR优化后，使用接种了不同生理比例精子：粘质沙雷氏菌（100:1、50:1、20:1、10:1、8:1、6:1、4:1、2:1、1:1和1:10）的公猪稀释精液评估检测灵敏度。将样品置于16°C下，在96小时内每24小时检测一次，在每个时间点使用商业试剂盒提取细菌DNA。作为最后一步，使用开发的技术对公猪稀释精液的随机样本进行粘质沙雷氏菌污染筛查。结果表明，这种PCR技术在检测不同接种比例以及公猪稀释精液的随机自然污染样本中的粘质沙雷氏菌时，灵敏度为90%，特异性为100%。总之，本研究报告了一种传统PCR技术，该技术能有效快速准确地检测公猪稀释精液中粘质沙雷氏菌的存在。
