Identification and quantification of Lactobacillus casei strain Shirota in human feces with strain-specific primers derived from randomly amplified polymorphic DNA
Identification and quantification of Lactobacillus casei strain Shirota in human feces with strain-specific primers derived from randomly amplified polymorphic DNA
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DOI：
10.1016/j.ijfoodmicro.2008.05.022
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发表时间：
2008-08-15
影响因子：
5.4
通讯作者：
Watanabe, Koichi
中科院分区：
文献类型：
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作者：
Fujimoto, Junji;Matsuki, Takahiro;Watanabe, Koichi
干酪乳杆菌代田菌株 (LcS) 多年来一直用于发酵乳制品的生产，是研究最深入的益生菌之一。为了评估 LcS 被摄入后在人体肠道中增殖的能力，我们开发了一种基于 PCR 的方法，使用源自随机扩增多态性 DNA (RAPD) 分析的 LcS 特异性引物组 (pLcS) 来识别和量化 LcS。我们证实了 pLcS 引物组在 167 种细菌菌株（57 种干酪乳杆菌菌株和 110 种通常从人类粪便中分离的其他细菌菌株）中具有高特异性。该方法识别 LcS 的能力与使用单克隆抗体的 ELISA 以及对从人类粪便培养的代表性菌落样本中进行的 RAPID 分析的能力相匹配。使用 pLcS 的定量 PCR (qPCR) 检测限为每克粪便 10(4.6)。 qPCR检测到的粪便中LcS数量与粪便样本中添加的LcS数量呈高度显着相关，在每克粪便10（4.6）至10（9.6）个范围内（r（2）=0.999，P < 0.001）。 14 名健康受试者每天摄入 10(11.0) CFU LcS，持续 7 天。通过 qPCR 在所有受试者的粪便样本中检测到 10(9.1 +/- 0.5) LcS g(-1) (平均值 +/- S.D.)，通过培养检测到 10(8.0 +/- 0.9) CFU g(-1)：这些值显着不同（P < 0.001，配对 t 检验）。受试者停止摄入 LcS 后，用这两种方法获得的粪便 LcS 计数每天都在减少。两种方法产生的值可能会有所不同，因为由于存在死亡的 LcS 细胞而导致 PCR 分析中的值被高估，或者由于使用选择性培养基而导致培养系统中的值被低估；然而，死亡的 LcS 细胞也可以作为免疫调节剂。我们证实，使用 LcS 特异性引物组的 qPCR 是一种快速、准确的方法，可用于测定粪便中 LcS 的总量，包括培养方法无法检测到的死亡或活性较低的细胞。 (C) 2008 Elsevier B.V. 保留所有权利。
Lactobacillus casei strain Shirota (LcS) has been used in the production of fermented milk products for many years and is one of the most intensively studied probiotics. To evaluate the ability of LcS to proliferate in human intestines after it has been ingested, we developed a PCR-based method to identify and quantify LcS using an LcS-specific primer set (pLcS) derived from a randomly amplified polymorphic DNA (RAPD) analysis. We confirmed the high specificity of the pLcS primer set in 167 bacterial strains (57 strains of L. casei and 110 other strains of bacteria commonly isolated from human feces). The method's ability to identify LcS matched that of an ELISA using a monoclonal antibody and a RAPID analysis in a representative sample of colonies cultured from human feces. The detection limit of quantitative PCR (qPCR) using pLcS was 10(4.6) per gram of feces. The number of LcS in feces detected with qPCR was highly and significantly correlated with the number of LcS added to fecal samples within the range of 10(4.6) to 10(9.6) per gram feces (r(2) =0.999, P < 0.001). After 14 healthy subjects ingested 10(11.0) CFU of LcS daily for 7 days. 10(9.1 +/- 0.5) LcS g(-1) (mean +/- S.D.) was detected in the fecal samples of all subjects by qPCR, and 10(8.0 +/- 0.9) CFU g(-1) was detected by culture: these values were significantly different (P < 0.001, paired t-test). After the subjects stopped ingesting LcS, fecal LcS counts obtained with both methods decreased daily. The values produced by the 2 methods might have differed because of an overestimation in the PCR analysis due to the presence of dead LcS cells or an underestimation in the culture system due to the use of selective culture media; however, dead LcS cells can also be beneficial as immunomodulators. We confirmed that qPCR with an LcS-specific primer set was a rapid and accurate method for determining the total amount of LcS in feces including dead or less active cells which could not be detected by culture method. (C) 2008 Elsevier B.V. All rights reserved.