Suzuki Kunio, Sakai D K
Eastern Hokkaido Inland Water Fisheries Section, The Hokkaido Fish Hatchery, Notorominato 1-1, Abashiri, Hokkaido 093-0131, Japan.
Dis Aquat Organ. 2007 Mar 13;74(3):209-23. doi: 10.3354/dao074209.
Quantification of msa gene mRNA of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), was investigated using reverse transcription followed by real-time PCR assay on R. salmoninarum in culture, and in experimentally challenged chum salmon Oncorhynchus keta fry kidney tissues (total of 70 samples) after intraperitoneal (i.p.) injection and bath infection. Correlations of msa gene mRNA concentrations with culturable cell concentrations (as colony forming units [CFU]), determined by drop-plate culture method on selective kidney disease medium (SKDM) agar through a 12 wk incubation time, and msa gene DNA concentrations by real-time PCR assay were examined. Furthermore, ovarian fluid samples from wild chum salmon adults with no clinical signs of disease were collected from 8 rivers and from clinically infected kokanee 0. nerka and masu salmon O. masou that were reared in 1 and 2 hatcheries, respectively (total of 414 samples). All samples were examined by nested PCR assay. Then, positive samples were examined by real-time PCR assays for mRNA and DNA; mRNA was detectable at 8 log units (5.0 x 101 to 5.0 x 10(9) copies p11(-1)) with high correlation (R2 = 0.999). The mRNA concentration correlated with CFU in kidney tissue from fish infected by i.p. injection (R2 = 0.924), by bath infection (R2 = 0.502) and in culture (R2 = 0.888). R. salmoninarum was detected and quantified by real-time PCR assay for mRNA in ovarian fluid samples in both subclinically infected chum salmon adults and clinically infected kokanee and masu salmon adults; detection rates ranged from 0 to 44.4% and concentrations ranged from 9.7 x 10(2) to 5.6 x 10(5) copies pl(-1). These results indicate that real-time PCR assay for the mRNA is a rapid, sensitive and reliable method to detect and quantify the viability of R. salmoninarum in kidney and ovarian fluid samples of salmonid fishes with both clinical and subclinical infection of the pathogen.
采用逆转录法，随后对培养的鲑肾杆菌以及腹腔注射和浸浴感染后实验性攻毒的大麻哈鱼幼鱼肾脏组织（共70个样本）进行实时PCR检测，以研究细菌性肾病（BKD）病原体鲑肾杆菌的msa基因mRNA的定量。通过在选择性肾病培养基（SKDM）琼脂上采用倾注平板培养法，经过12周的培养时间，确定可培养细胞浓度（以菌落形成单位[CFU]计），并通过实时PCR检测msa基因DNA浓度，研究了msa基因mRNA浓度与它们的相关性。此外，分别从8条河流中采集了无疾病临床症状的野生大麻哈鱼成体的卵巢液样本，以及从分别饲养在1个和2个孵化场的临床感染的红大麻哈鱼和马苏大麻哈鱼中采集了卵巢液样本（共414个样本）。所有样本均通过巢式PCR检测。然后，对阳性样本进行mRNA和DNA的实时PCR检测；mRNA在8个对数单位（5.0×10¹至5.0×10⁹拷贝μl⁻¹）可检测到，相关性很高（R² = 0.999）。mRNA浓度与腹腔注射感染（R² = 0.924）、浸浴感染（R² = 0.502）以及培养（R² = 0.888）的鱼类肾脏组织中的CFU相关。通过实时PCR检测mRNA，在亚临床感染的大麻哈鱼成体以及临床感染的红大麻哈鱼和马苏大麻哈鱼成体的卵巢液样本中均检测到并定量了鲑肾杆菌；检测率范围为0至44.4%，浓度范围为9.7×10²至5.6×10⁵拷贝μl⁻¹。这些结果表明，针对mRNA的实时PCR检测是一种快速、灵敏且可靠的方法，可用于检测和定量患有该病原体临床和亚临床感染的鲑科鱼类肾脏和卵巢液样本中鲑肾杆菌的活力。
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