基于16S rDNA的基因测序在腹透相关腹膜炎早期诊断中的应用

发布时间：2018-02-27 20:59

本文关键词： 腹膜透析腹膜炎 16S rDNA PCR 基因测序序列比对　出处：《浙江大学》2015年硕士论文　论文类型：学位论文

【摘要】：研究背景：腹膜透析(peritoneal dialysis,以下简称PD)是终末期肾病(end-stage renal disease, ESRD)的主要肾脏替代治疗方法之一。腹透相关腹膜炎是腹膜透析最常见的感染性并发症,严重者可引起腹透技术失败,甚至死亡。细菌学培养是腹透相关腹膜炎诊断的可靠依据。然而,传统的细菌培养有周期长、阳性率偏低、易受抗生素使用影响等不足。因此,寻找能快速、有效鉴定菌种的新方法具有重要意义。近年来,以16S rDNA为基础的分子生物学技术因快速、灵敏、准确等优点在各类疾病的菌种鉴定中尤受青睐。16S rDNA是原核生物核糖体中16S rRNA对应的基因序列,既有高度保守性,又具有一定变异性。利用保守区域设计通用引物或特异探针,再利用可变区的差异鉴别菌种,这就是此类分子生物技术的基本原理。研究目的：探讨基于16S rDNA的基因测序在腹透相关腹膜炎早期诊断中的临床应用价值。实验方法：本研究选取69例腹透相关腹膜炎的腹膜透析透出液标本作为研究对象,选取16S rDNA保守区的通用引物27F/1492R进行PCR扩增,对扩增所得的目的基因进行测序及序列比对确定细菌,最后与常规培养法相比较。此外,取18例腹透病人的正常腹膜透析透出液作为对照组,予上述测序方法及常规培养鉴定有无细菌。结果：在69例腹透相关腹膜炎的腹膜透析透出液标本中,测序法鉴定病原菌的阳性率(71%,49/69)虽然高于常规培养法(63.8%,44/69),但未达统计学显著性差异(P0.05)。在44例培养阳性的标本中,测序法的一致性为72.7%(32/44)。在25例培养阴性的标本中,测序法鉴定出8例菌株,追踪病史可知其中3例患者在前一次/后一次腹膜炎感染时常规培养证实有相同的病原菌感染,剩余5例患者有培养阴性的腹膜炎记录或未再发生腹膜炎。在有近期抗生素使用史的18例标本中,测序法的阳性率(61.1%、11/18)高于常规培养法(50%,9/18),但未达统计学显著性差异(P0.05)。作为对照组的18例腹膜透析透出液经测序法和细菌培养法,均无阳性结果。结论：基于16S rDNA的测序法的阳性率高于常规培养法,虽未达到明显统计学差异,但测序法具有耗时短、花费少、操作简单、不受抗生素影响等优点,可作为常规病原学培养的有益补充,具有早期、快速诊断腹透相关腹膜炎的重要的临床应用价值。
[Abstract]:Background: peritoneal dialysis (PDD) is one of the main methods of renal replacement therapy for end-stage renal disease (ESRD). Peritoneal dialysis associated peritonitis is the most common infectious complication of peritoneal dialysis. Even death. Bacteriological culture is a reliable basis for diagnosis of peritonitis associated with peritoneal dialysis. However, the traditional bacterial culture has a long period, low positive rate, vulnerable to antibiotic use and so on. In recent years, the molecular biological techniques based on 16s rDNA have been proved to be rapid and sensitive. In the identification of various diseases, 16s rDNA is the gene sequence corresponding to 16s rRNA in prokaryotes, which is both highly conserved and variable. This is the basic principle of this kind of molecular biotechnology. Objective: to evaluate the clinical value of 16s rDNA gene sequencing in early diagnosis of peritoneal dialysis associated peritonitis. Methods: in this study, 69 peritoneal dialysate samples from patients with peritoneal dialysis associated peritonitis were selected for PCR amplification. The 16s rDNA conserved primer 27F / 1492R was used for PCR amplification. The amplified target gene was sequenced and sequenced to determine the bacteria, and compared with the conventional culture method. In addition, the normal peritoneal dialysis solution from 18 patients with abdominal dialysis was taken as the control group. The above sequencing method and routine culture were used to identify the presence of bacteria. Results: in 69 peritoneal dialysis effusion specimens of peritoneal dialysis associated peritonitis, the positive rate of pathogenic bacteria identified by sequencing method was higher than that by conventional culture method (63.8% / 69%), but the difference was not statistically significant (P 0.05). The consistency of sequencing was 72.7 / 44. In 25 culture-negative specimens, 8 strains were identified by sequencing, and 3 of them were confirmed to have the same pathogen by routine culture during previous / subsequent peritonitis infection. The remaining 5 patients had a culture negative peritonitis record or no recurrence of peritonitis. In 18 specimens with a history of recent antibiotic use, The positive rate of sequencing was higher than that of conventional culture method (50 / 18 / 18), but the difference was not statistically significant (P 0.05). No positive results were found in 18 cases of peritoneal dialysis dialysate as control group by sequencing and bacterial culture. Conclusion: the positive rate of 16s rDNA sequencing method is higher than that of conventional culture method. Although there is no significant statistical difference, sequencing method has the advantages of short time consuming, low cost, simple operation, and not affected by antibiotics. It can be used as a useful supplement for routine etiological culture and has important clinical application value in early and rapid diagnosis of peritoneal dialysis associated peritonitis.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R692;R572.2

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