Kanto Chemical Co., Inc.
Jpn J Infect Dis. 2013;66(5):383-90. doi: 10.7883/yoken.66.383.
In this study, a specific quantitative PCR system for the detection and identification of Prototheca zopfii genotypes was developed using a TaqMan(®) MGB probe and ResoLight dye. The P. zopfii-specific primers 18PZF1 and 18PZR1 were generated on the basis of the alignment of the small subunit ribosomal DNA domain base sequences of the genera Chlorella and Prototheca obtained from DDBJ/EMBL/GenBank, and the TaqMan(®) MGB probe PZP1 was designed corresponding to this amplification region. Analysis of the melting curves of the amplicons using ResoLight dye was able to differentiate between P. zopfii genotypes 1 and 2. The specificity of this detection system was examined using strains from a culture collection (28 strains) and clinical isolates (140 strains). The TaqMan(®) MGB probe amplicon was detected only in reference strains of P. zopfii (n = 12) and clinical isolates (n = 135). Ninety-two clinical specimens from cows with mastitis (36 samples) and healthy controls (56 samples) were also tested. All isolates from milk samples (n = 92) and clinical isolates (n = 135) were identified as P. zopfii genotype 2.
在这项研究中，使用 TaqMan(®) MGB 探针和 ResoLight 染料开发了一种用于检测和鉴定 Zopf 小球藻基因型的特定定量 PCR 系统。基于从 DDBJ/EMBL/GenBank 获得的小球藻属和 Prototheca 属的小亚基核糖体 DNA 结构域碱基序列的比对，生成了 P. zopfii 特异性引物 18PZF1 和 18PZR1，并且设计了与该扩增区域相对应的 TaqMan(®) MGB 探针 PZP1。使用 ResoLight 染料分析扩增产物的熔解曲线能够区分 P. zopfii 基因型 1 和 2。使用来自培养物收集（28 株）和临床分离株（140 株）的菌株检查了该检测系统的特异性。TaqMan(®) MGB 探针扩增子仅在 P. zopfii 的参考菌株（n = 12）和临床分离株（n = 135）中检测到。还测试了来自乳腺炎奶牛的 92 份临床标本（36 个样本）和健康对照（56 个样本）。从牛奶样本（n = 92）和临床分离株（n = 135）中分离的所有菌株均鉴定为 P. zopfii 基因型 2。
