本文關(guān)鍵詞： 腹膜透析 腹膜炎 16S rDNA PCR 基因測序 序列比對　出處：《浙江大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景：腹膜透析(peritoneal dialysis,以下簡稱PD)是終末期腎病(end-stage renal disease, ESRD)的主要腎臟替代治療方法之一。腹透相關(guān)腹膜炎是腹膜透析最常見的感染性并發(fā)癥,嚴(yán)重者可引起腹透技術(shù)失敗,甚至死亡。細(xì)菌學(xué)培養(yǎng)是腹透相關(guān)腹膜炎診斷的可靠依據(jù)。然而,傳統(tǒng)的細(xì)菌培養(yǎng)有周期長、陽性率偏低、易受抗生素使用影響等不足。因此,尋找能快速、有效鑒定菌種的新方法具有重要意義。近年來,以16S rDNA為基礎(chǔ)的分子生物學(xué)技術(shù)因快速、靈敏、準(zhǔn)確等優(yōu)點(diǎn)在各類疾病的菌種鑒定中尤受青睞。16S rDNA是原核生物核糖體中16S rRNA對應(yīng)的基因序列,既有高度保守性,又具有一定變異性。利用保守區(qū)域設(shè)計(jì)通用引物或特異探針,再利用可變區(qū)的差異鑒別菌種,這就是此類分子生物技術(shù)的基本原理。 研究目的：探討基于16S rDNA的基因測序在腹透相關(guān)腹膜炎早期診斷中的臨床應(yīng)用價(jià)值。 實(shí)驗(yàn)方法：本研究選取69例腹透相關(guān)腹膜炎的腹膜透析透出液標(biāo)本作為研究對象,選取16S rDNA保守區(qū)的通用引物27F/1492R進(jìn)行PCR擴(kuò)增,對擴(kuò)增所得的目的基因進(jìn)行測序及序列比對確定細(xì)菌,最后與常規(guī)培養(yǎng)法相比較。此外,取18例腹透病人的正常腹膜透析透出液作為對照組,予上述測序方法及常規(guī)培養(yǎng)鑒定有無細(xì)菌。 結(jié)果：在69例腹透相關(guān)腹膜炎的腹膜透析透出液標(biāo)本中,測序法鑒定病原菌的陽性率(71%,49/69)雖然高于常規(guī)培養(yǎng)法(63.8%,44/69),但未達(dá)統(tǒng)計(jì)學(xué)顯著性差異(P0.05)。在44例培養(yǎng)陽性的標(biāo)本中,測序法的一致性為72.7%(32/44)。在25例培養(yǎng)陰性的標(biāo)本中,測序法鑒定出8例菌株,追蹤病史可知其中3例患者在前一次/后一次腹膜炎感染時(shí)常規(guī)培養(yǎng)證實(shí)有相同的病原菌感染,剩余5例患者有培養(yǎng)陰性的腹膜炎記錄或未再發(fā)生腹膜炎。在有近期抗生素使用史的18例標(biāo)本中,測序法的陽性率(61.1%、11/18)高于常規(guī)培養(yǎng)法(50%,9/18),但未達(dá)統(tǒng)計(jì)學(xué)顯著性差異(P0.05)。作為對照組的18例腹膜透析透出液經(jīng)測序法和細(xì)菌培養(yǎng)法,均無陽性結(jié)果。 結(jié)論：基于16S rDNA的測序法的陽性率高于常規(guī)培養(yǎng)法,雖未達(dá)到明顯統(tǒng)計(jì)學(xué)差異,但測序法具有耗時(shí)短、花費(fèi)少、操作簡單、不受抗生素影響等優(yōu)點(diǎn),可作為常規(guī)病原學(xué)培養(yǎng)的有益補(bǔ)充,具有早期、快速診斷腹透相關(guān)腹膜炎的重要的臨床應(yīng)用價(jià)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R692;R572.2

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相關(guān)期刊論文 前2條

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本文編號：1544294