Okeke C N, Tsuboi R, Kawai M, Yamazaki M, Reangchainam S, Ogawa H
Department of Dermatology, Juntendo University School of Medicine, Tokyo, Japan.
J Invest Dermatol. 2000 Jan;114(1):95-100. doi: 10.1046/j.1523-1747.2000.00845.x.
The presence of viable cells of Candida albicans, in broth or in a reconstructed living skin equivalent, was determined by the detection of amplicons of partial mRNA sequences of the genes encoding fungal actin (ACT1) and secreted aspartyl proteinase 2 (SAP2). The mRNA of both genes were amplified by reverse transcription-3' rapid amplification of cDNA ends-nested polymerase chain reaction. Single bands of ACT1 (315 bp) and SAP2 (162 bp) mRNA were amplified from total RNA extracts of C. albicans grown in yeast carbon base-albumin broth or in living skin equivalent tissue; only the former was amplified from Sabouraud broth-grown organisms. Primer pairs targeted for ACT1 and SAP2 were Candida genus-specific and C. albicans-specific, respectively. The sensitivity limits of the assay were 100 fg of total RNA or 10 cells of C. albicans, by ethidium bromide staining. When C. albicans-infected living skin equivalent was exposed to amorolfine, amplicons of ACT1 and SAP2 mRNA were not detected in total RNA extracts. Non-amplification of the mRNA correlated with the absence of C. albicans growth in Sabouraud agar cultures of living skin equivalent samples. Reverse transcription-3' rapid amplification of cDNA ends-nested polymerase chain reaction of the mRNA encoding specific proteins of an organism has potential application in determining the viability of the organism in tissue, thus monitoring the efficacy of an antimicrobial therapy, and in detecting mRNA expressed in very little amounts in tissue.
通过检测编码真菌肌动蛋白（ACT1）和分泌天冬氨酸蛋白酶2（SAP2）的基因部分mRNA序列的扩增子，来确定白色念珠菌在肉汤或重建的活皮肤替代物中的活细胞存在情况。这两个基因的mRNA通过逆转录-3' cDNA末端快速扩增-巢式聚合酶链反应进行扩增。从在酵母碳源-白蛋白肉汤或活皮肤替代物组织中生长的白色念珠菌的总RNA提取物中扩增出ACT1（315 bp）和SAP2（162 bp）mRNA的单一条带；仅从在沙氏肉汤中生长的生物体中扩增出前者。针对ACT1和SAP2的引物对分别是念珠菌属特异性和白色念珠菌特异性的。通过溴化乙锭染色，该检测方法的灵敏度极限为100 fg总RNA或10个白色念珠菌细胞。当感染白色念珠菌的活皮肤替代物暴露于阿莫罗芬时，在总RNA提取物中未检测到ACT1和SAP2 mRNA的扩增子。mRNA未扩增与活皮肤替代物样品的沙氏琼脂培养物中白色念珠菌生长的缺乏相关。对生物体特定蛋白质编码mRNA进行逆转录-3' cDNA末端快速扩增-巢式聚合酶链反应在确定生物体在组织中的活力、监测抗菌治疗效果以及检测组织中少量表达mRNA方面具有潜在应用价值。
