Aguilera-Arreola Ma Guadalupe, González-Cardel Ana María, Tenorio Alfonso Méndez, Curiel-Quesada Everardo, Castro-Escarpulli Graciela
Medical bacteriology, Department of Microbiology, Escuela Nacional de Ciencias Biológicas of Instituto Politécnico Nacional (ENCB-IPN), Esq, Prol, Carpio y Plan de Ayala s/n Col, Santo Tomás, Del, Miguel Hidalgo CP 11340, Mexico DF.
BMC Res Notes. 2014 Jul 6;7:433. doi: 10.1186/1756-0500-7-433.
Although sophisticated methodologies are available, the use of endpoint polymerase chain reaction (PCR) to detect 16S rDNA genes remains a good approach for estimating the incidence and prevalence of specific infections and for monitoring infections. Considering the importance of the early diagnosis of sexually transmitted infections (STIs), the development of a sensitive and affordable method for identifying pathogens in clinical samples is needed. Highly specific and efficient primers for a multiplex polymerase chain reaction (m-PCR) system were designed in silico to detect the 16S rDNA genes of four bacteria that cause genital infections, and the PCR method was developed.
The Genosensor Probe Designer (GPD) (version 1.0a) software was initially used to design highly specific and efficient primers for in-house m-PCR. Single-locus PCR reactions were performed and standardised, and then primers for each locus in turn were added individually in subsequent amplifications until m-PCR was achieved. Amplicons of the expected size were obtained from each of the four bacterial gene fragments. Finally, the analytical specificity and limits of detection were tested.
Because they did not amplify any product from non-STI tested species, the primers were specific. The detection limits for the Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum primer sets were 5.12 × 10(5), 3.9 × 10(3), 61.19 × 10(6) and 6.37 × 10(5) copies of a DNA template, respectively.
The methodology designed and standardised here could be applied satisfactorily for the simultaneous or individual detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum. This method is at least as efficient as other previously described methods; however, this method is more affordable for low-income countries.
尽管有复杂的方法可用，但使用终点聚合酶链反应（PCR）检测16S rDNA基因仍是估计特定感染的发病率和患病率以及监测感染情况的良好方法。考虑到性传播感染（STIs）早期诊断的重要性，需要开发一种灵敏且经济实惠的方法来鉴定临床样本中的病原体。通过计算机辅助设计了用于多重聚合酶链反应（m-PCR）系统的高度特异性和高效的引物，以检测四种引起生殖器感染的细菌的16S rDNA基因，并开发了PCR方法。
最初使用基因传感器探针设计软件（GPD）（版本1.0a）设计用于内部m-PCR的高度特异性和高效的引物。进行单基因座PCR反应并标准化，然后在随后的扩增中依次单独添加每个基因座的引物，直至实现m-PCR。从四个细菌基因片段中的每一个都获得了预期大小的扩增子。最后，测试了分析特异性和检测限。
由于这些引物未从非性传播感染测试物种中扩增出任何产物，因此具有特异性。沙眼衣原体、淋病奈瑟菌、人型支原体和解脲脲原体引物组的检测限分别为5.12×10⁵、3.9×10³、61.19×10⁶和6.37×10⁵个DNA模板拷贝。
此处设计和标准化的方法可令人满意地用于同时或单独检测沙眼衣原体、淋病奈瑟菌、人型支原体和解脲脲原体。该方法至少与其他先前描述的方法一样有效；然而，对于低收入国家来说，该方法更经济实惠。
