Kim Yoonjung, Kim Juwon, Lee Kyung-A
Clin Lab. 2015;61(11):1749-54. doi: 10.7754/clin.lab.2015.150413.
Most organisms that cause sexual transmitted diseases (STDs) are fastidious pathogens that are difficult to detect with conventional microbiological methods and the proportions of multiple infections were noted up to 39.3% among the STI-positive subjects. However, only a few multiplex PCR and multiplex real-time PCR tests that can screen more than six microorganisms that cause STDs have been assessed.
A total of 114 endocervical swabs (ThinPrep PAPTEST PreservCyt Solution, Hologic Inc., Marlborough, MA, USA) were collected from healthy Korean women. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), and Trichomonas vaginalis (TV) were detected by uniplex PCR with Seeplex kits and by multiplex real-time PCR with Real-Q Kits (Biosewoom Inc., Seoul, Korea). To evaluate analytical sensitivity, plasmids containing target genes from CT, NG, MG, MH, UU, and TV were serially diluted five times with saline buffer and replicated eight times per dilution.
Real-Q STIs Kit assays showed 100% sensitivity for detecting MH, MG, CT, TV, NG and 94.1% sensitivity for detecting UU. In addition, it showed 100% specificity for UU, MH, MG, CT, TV, and NG. The analytic sensitivity of UU (95% probit = 17.3 copy/μL, 95% CI = 11.6 to 138.6) and MH (95% probit = 30.9 copy/μL, 95% CI = 20.6 to 169.9) was relatively lower than for others pathogens. Thus, the cutoff Ct value of < 45 for UU and MH and a cutoff Ct value of < 38 for CT, MH, NG, TV could minimize differences in detection limit among the six STIs (95% probit values = 5.3 to 14.6) and to optimize overall diagnostic performance.
For medical applications of a multiplex real-time PCR assay, one kind of cutoff value, which is according to manufacturer's instructions, was generally used without the consideration of lowest actual detectable concentration of each target substance. However, analytical performance at the low concentration limit often defines the ability of the test to diagnose disease and determine treatment endpoints. Therefore, suitable cutoffs for negative or positive screens by multiplex real-time PCR should be evaluated for accurate diagnosis.
大多数引起性传播疾病（STD）的病原体是苛求菌，难以用传统微生物学方法检测，并且在性传播感染阳性受试者中多重感染比例高达39.3%。然而，仅有少数几种可筛查六种以上引起STD的微生物的多重PCR和多重实时PCR检测方法得到评估。
从健康韩国女性中收集了114份宫颈拭子（ThinPrep PAPTEST PreservCyt Solution，美国马萨诸塞州马尔伯勒市Hologic公司）。采用Seeplex试剂盒通过单重PCR以及采用Real-Q试剂盒（韩国首尔市Biosewoom公司）通过多重实时PCR检测沙眼衣原体（CT）、淋病奈瑟菌（NG）、生殖支原体（MG）、人型支原体（MH）、解脲脲原体（UU）和阴道毛滴虫（TV）。为评估分析灵敏度，将含有CT、NG、MG、MH、UU和TV靶基因的质粒用生理盐水缓冲液连续稀释5次，每次稀释重复8次。
Real-Q STIs试剂盒检测对MH、MG、CT、TV、NG的灵敏度为100%，对UU的灵敏度为94.1%。此外，对UU、MH、MG、CT、TV和NG的特异性均为100%。UU（95%概率单位=17.3拷贝/μL，95%置信区间=11.6至138.6）和MH（95%概率单位=30.9拷贝/μL，95%置信区间=20.6至169.9）的分析灵敏度相对低于其他病原体。因此，UU和MH的截断Ct值<45，CT、MH、NG、TV的截断Ct值<38，可使六种性传播感染的检测限差异最小化（95%概率单位值=5.3至14.6）并优化总体诊断性能。
对于多重实时PCR检测的医学应用，通常按照制造商说明使用一种截断值，而未考虑每种靶物质的实际最低可检测浓度。然而，低浓度极限下的分析性能往往决定了检测诊断疾病和确定治疗终点的能力。因此，应评估多重实时PCR阴性或阳性筛查的合适截断值以进行准确诊断。
