Ryan Gavin J, Shapiro Howard M, Lenaerts Anne J
Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
The Center for Microbial Cytometry and Howard M. Shapiro, M.D., P.C., 283 Highland Avenue, West Newton, MA 02465-2513, USA.
Tuberculosis (Edinb). 2014 Sep;94(5):511-8. doi: 10.1016/j.tube.2014.07.004. Epub 2014 Jul 28.
Acid fast staining of sputum smears by microscopy remains the prevalent method for detecting Mycobacterium tuberculosis. The sensitivity of microscopy using acid fast stains requires 10(4) bacilli per ml of sputum. Although fluorescent acid fast stains, such as Auramine-O, show improved sensitivity, almost half of culture-positive TB cases are currently estimated to remain smear-negative. These current diagnosis problems provide impetus for improving staining procedures. We evaluated a novel fluorescent acid-fast staining approach using the nucleic acid-binding dye SYBR(®) Gold on mycobacterial in vitro cultures. The SYBR(®) Gold stain detected 99% of MTB in both actively replicating aerobic and non-replicating hypoxic cultures. Transmission light microscopy with Ziehl-Neelsen fuchsin, and fluorescence microscopy with Auramine-O or Auramine-rhodamine detected only 54%-86% of MTB bacilli. SYBR(®) Gold fluoresces more intensely than Auramine-O, and is highly resistant to fading. The signal to noise ratio is exceptionally high due to a >1000-fold enhanced fluorescence after binding to DNA/RNA, thereby reducing most background fluorescence. Although cost and stability of the dye may perhaps limit its clinical use at this time, these results warrant further research into more nucleic acid dye variants. In the meantime, SYBR(®) Gold staining shows great promise for use in numerous research applications.
通过显微镜对痰涂片进行抗酸染色仍然是检测结核分枝杆菌的常用方法。使用抗酸染色的显微镜检测灵敏度要求每毫升痰中有10⁴条杆菌。尽管诸如金胺O等荧光抗酸染色显示出更高的灵敏度，但据估计目前几乎一半培养阳性的结核病病例痰涂片仍为阴性。当前的这些诊断问题推动了对染色程序的改进。我们评估了一种使用核酸结合染料SYBR® Gold对分枝杆菌体外培养物进行新型荧光抗酸染色的方法。SYBR® Gold染色在活跃复制的需氧培养物和非复制性缺氧培养物中均检测到99%的结核分枝杆菌。用齐-尼氏石炭酸复红进行的透射光显微镜检查，以及用金胺O或金胺-罗丹明进行的荧光显微镜检查仅检测到54%-86%的结核杆菌。SYBR® Gold的荧光比金胺O更强，且高度抗褪色。由于与DNA/RNA结合后荧光增强超过1000倍，信噪比极高，从而减少了大部分背景荧光。尽管目前该染料的成本和稳定性可能会限制其临床应用，但这些结果值得对更多核酸染料变体进行进一步研究。与此同时，SYBR® Gold染色在众多研究应用中显示出巨大的应用前景。
