A novel peptide nucleic acid- and loop-mediated isothermal amplification assay for the detection of mutations in the 23S rRNA gene of Treponema pallidum
A novel peptide nucleic acid- and loop-mediated isothermal amplification assay for the detection of mutations in the 23S rRNA gene of Treponema pallidum
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DOI：
10.1099/jmm.0.001275
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发表时间：
2020-01-01
影响因子：
3
通讯作者：
Maeda, Takuya
中科院分区：
文献类型：
--
作者：
Tarumoto, Norihito;Imai, Kazuo;Maeda, Takuya
导言。大环内酯类药物可能是治疗梅毒螺旋体感染的潜在替代疗法；然而，具有大环内酯类抗药性的梅毒螺旋体正在世界范围内迅速传播。由于梅毒螺旋体不能在体外培养，目前还没有梅毒血清学试验的替代方法可以用来评估治疗效果。在本研究中，我们建立了一种快速鉴定梅毒螺旋体和确认大环内酯类抗药性的方法--环介导肽核酸等温扩增(LAMP)方法。设计了一组跨越23S rRNA中2058和2059位核苷酸的LAMP引物。还设计了与野生型序列(A2058/A2059)互补的PNA夹持探针，并定位为干扰反向内引物3‘端的退火和伴随的延伸。在LAMP检测之前，对疑似梅毒病变的拭子样本进行煮沸以提取DNA。该方法的检测下限为1.0×10(1)拷贝/反应，对38种病原菌具有较高的特异性。在4 mU M PNA探针存在下，LAMP利用含有互补突变序列的质粒(A2058G或A2059G)扩增高达1.0x10(1)拷贝/反应，而对于野生型序列扩增被完全阻断，浓度高达1.0x10(3)拷贝/反应。对66例阳性的临床标本，LAMP检测总阳性率为93.9%(62/66)。经PNA介导的LAMP分析，53份突变样本全部扩增成功，9份WT样本全部扩增不完全。我们开发了一种PNA介导的LAMP方法，使我们能够通过不依赖培养的方法快速鉴定梅毒螺旋体并确定其对大环内酯类药物的敏感性。
Introduction. Macrolides could be a potential alternative treatment for Treponema pallidum infections in patients; however, macrolide-resistant T. pallidum is spreading rapidly worldwide.Hypothesis/Gap Statement. There are presently no alternatives to serological tests for syphilis that can be used to evaluate therapeutic effects due to the fact that T. pallidum cannot be cultured in vitro.Aim. In this study, we constructed a method for rapidly identifying T. pallidum and confirming macrolide resistance by using loop-mediated isothermal amplification (LAMP) with peptide nucleic acids (PNAs).Methodology. A set of LAMP primers was designed to span nucleotide positions 2058 and 2059 in 23S rRNA. A PNA clamping probe was also designed to be complementary to the wild type sequence (A2058/A2059) and positioned to interfere with both the annealing of the 3' end of the backward inner primer and the concomitant extension. Prior to the LAMP assay, swab samples from suspected syphilitic lesions were boiled for DNA extraction.Results. The assay had an equivalent detection limit of 1.0x10(1) copies/reaction and showed specificity against 38 pathogens. In the presence of a 4 mu M PNA probe, LAMP amplified up to 1.0x10(1) copies/reaction using plasmids harbouring the complementary mutant sequences (A2058G or A2059G), whereas amplification was completely blocked for the wild type sequence up to a concentration of 1.0x10(3) copies/reaction. For the 66 PCR-positive clinical specimens, the overall detection rate via LAMP was 93.9 % (62/66). Amplification was successful for all 53 mutant samples and was incomplete for all nine WT samples by the PNA-mediated LAMP assays.Conclusion. We developed a PNA-mediated LAMP method that enabled us to rapidly identify T. pallidum and determine its macrolide susceptibility via a culture-independent protocol.