Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma, Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases.
Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma, Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases.
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DOI：
10.1371/journal.pone.0129032
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Kitajima I
中科院分区：
文献类型：
--
作者：
Ueno T;Niimi H;Yoneda N;Yoneda S;Mori M;Tabata H;Minami H;Saito S;Kitajima I
羊膜腔感染一直被认为是早产的主要原因。微生物培养是检测羊水内感染的金标准，但需要几天时间，而且羊水中的许多细菌很难培养。我们开发了一种新的巢式PCR为基础的检测支原体，脲原体，其他细菌和真菌在羊水样本采集三小时内。为了检测原核生物，将不受细菌DNA污染的真核生物制造的热稳定DNA聚合酶与细菌通用引物组合使用。相比之下，为了检测真核生物，常规细菌制造的热稳定DNA聚合酶与真菌通用引物组合使用。为了评估PCR检测的有效性，我们使用300份羊水样本比较了PCR和传统培养的结果。根据检测水平（阳性和阴性），支原体93.3%（280/300）、支原体94.3%（283/300）、其他细菌89.3%（268/300）和真菌99.7%（299/300）与培养结果相符。同时，关于支原体和脲原体以外的细菌检测，228份样本根据PCR方法为阴性，其中98.2%（224/228）的样本根据培养方法也为阴性。利用设计的引物组，支原体、支原体和/或其他细菌的混合感染的羊水可以清楚地区分。此外，我们还尝试比较28例混合感染的羊水样本的相对丰度，并通过比较定量实时PCR的Ct值来判断优势。我们开发了一种新的PCR方法，用于快速检测羊水样本中的支原体、支原体、其他细菌和真菌。该测定也可用于准确诊断样品中是否存在细菌。我们相信，这种检测将积极有助于治疗羊膜内感染和预防早产。
Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate. We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made thermostable DNA polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made thermostable DNA polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples. Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR. We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and the prevention of preterm delivery.
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影响因子：
36.8
作者：
CASSELL, GH;WAITES, KB;HARASAWA, R
通讯作者：
HARASAWA, R
DOI：
10.1902/annals.1998.3.1.222
发表时间：
1998-07-01
期刊：
Annals of periodontology
影响因子：
--
作者：
Hill, G B
通讯作者：
Hill, G B
影响因子：
3.3
作者：
RAND, KH;HOUCK, H
通讯作者：
HOUCK, H
影响因子：
7.2
作者：
Kundsin, RB;Leviton, A;Pollin, SA
通讯作者：
Pollin, SA
DOI：
10.1016/j.ejogrb.2006.09.013
发表时间：
2007-09-01
影响因子：
2.6
作者：
Kirchner, Lieselotte;Helmer, Hanns;Zaknun, Daniela
通讯作者：
Zaknun, Daniela