Edberg Andreas, Aronsson Fredrik, Johansson Eva, Wikander Elisabeth, Ahlqvist Thomas, Fredlund Hans
Department of Chemistry and Biomedical Sciences, Karlstad University, SE-651 88 Karlstad, Sweden.
Department of Clinical Microbiology, Central Hospital, SE-651 85 Karlstad, Sweden.
J Med Microbiol. 2009 Jan;58(Pt 1):117-120. doi: 10.1099/jmm.0.003681-0.
The aim of this study was to determine whether a patient's endocervical swab specimen can be transported in first void urine (FVU) as combined specimens for the detection of Mycoplasma genitalium by real-time PCR. The study also compared two different DNA extraction methods for observation of possible PCR inhibition. Three specimens, one endocervical swab specimen transported in 2-SP medium, one endocervical swab specimen transported in FVU and a FVU specimen, were collected from 329 women. All sample types underwent manual DNA extraction whereas in the DNA extraction study, 329 endocervical swab specimens transported in FVU were subjected to both manual Chelex and automated BioRobot M48 DNA extraction. A total of 100 endocervical swab specimens transported in FVU from patients PCR-negative for M. genitalium in the study were used in the PCR inhibition analysis. M. genitalium was detected in 25/329 (7.6 %) women. The endocervical swab specimens transported in 2-SP medium and transported in FVU were positive for M. genitalium in 17/25 (68 %) and 24/25 (96 %) women, respectively. The FVU specimens alone were positive for M. genitalium in 22/25 (88 %) women. In the DNA extraction study, M. genitalium DNA was detected in 24/329 (7.3 %) and 28/329 (8.5 %) of endocervical swab specimens transported in FVU subjected to manual Chelex extraction and automated BioRobot M48 extraction, respectively. Partial PCR inhibition was detected in 6 % of samples subjected to manual Chelex extraction whereas no inhibition was detected with the automated BioRobot M48 extraction. Thus endocervical swab specimens transported in FVU demonstrate higher sensitivity than FVU specimens only and have considerably increased sensitivity compared with endocervical swab specimens transported in 2-SP medium for detection of M. genitalium DNA. Moreover, automated BioRobot M48 extraction was shown to be superior to a crude manual Chelex extraction, leaving no PCR inhibition and giving a slightly higher DNA yield and/or better sensitivity.
本研究的目的是确定患者的宫颈拭子标本是否可以与首次晨尿(FVU)混合作为联合标本进行运输,以便通过实时PCR检测生殖支原体。该研究还比较了两种不同的DNA提取方法,以观察是否存在可能的PCR抑制作用。从329名女性中收集了三种标本,一种是在2-SP培养基中运输的宫颈拭子标本,一种是在FVU中运输的宫颈拭子标本,还有一种是FVU标本。所有样本类型均进行手动DNA提取,而在DNA提取研究中,对329份在FVU中运输的宫颈拭子标本同时进行了手动Chelex提取和自动化BioRobot M48 DNA提取。在该研究中,从生殖支原体PCR阴性的患者中选取了100份在FVU中运输的宫颈拭子标本用于PCR抑制分析。在329名女性中,有25名(7.6%)检测到生殖支原体。在2-SP培养基中运输的宫颈拭子标本和在FVU中运输的宫颈拭子标本中,分别有17/25(68%)和24/25(96%)的女性生殖支原体呈阳性。仅FVU标本中,有22/25(88%)的女性生殖支原体呈阳性。在DNA提取研究中,在分别采用手动Chelex提取和自动化BioRobot M48提取的FVU运输的宫颈拭子标本中,生殖支原体DNA的检测率分别为24/329(7.3%)和28/329(8.5%)。在采用手动Chelex提取的样本中,有6%检测到部分PCR抑制,而采用自动化BioRobot M48提取未检测到抑制。因此,在FVU中运输的宫颈拭子标本显示出比仅FVU标本更高的灵敏度,并且与在2-SP培养基中运输的宫颈拭子标本相比,在检测生殖支原体DNA时灵敏度显著提高。此外,自动化BioRobot M48提取显示优于粗制的手动Chelex提取,不存在PCR抑制,DNA产量略高和/或灵敏度更好。