【摘要】：多重PCR檢測(cè)腸源性膿毒癥常見(jiàn)致病菌的方法構(gòu)建及初步臨床應(yīng)用目的構(gòu)建檢測(cè)腸源性膿毒癥常見(jiàn)病原菌的快速的、可靠的多重PCR反應(yīng)體系,并初步探索其臨床價(jià)值。方法根據(jù)10種腸源性膿毒癥常見(jiàn)致病菌(以下簡(jiǎn)稱目標(biāo)細(xì)菌)的16sRNA特異性片段設(shè)計(jì)互補(bǔ)序列的引物和探針。優(yōu)化多重PCR反應(yīng)體系并進(jìn)行特異性實(shí)驗(yàn)及濃度梯度敏感性實(shí)驗(yàn)。將42例腸源性膿毒癥患者隨機(jī)分為多重PCR檢測(cè)指導(dǎo)治療組(以下簡(jiǎn)稱多重PCR組)和經(jīng)驗(yàn)用藥及血培養(yǎng)指導(dǎo)組(以下簡(jiǎn)稱對(duì)照組),觀察兩組患者抗菌藥物使用時(shí)間、抗菌藥物總費(fèi)用、總住院費(fèi)用、SIRS持續(xù)時(shí)間以及各種并發(fā)癥發(fā)生率和死亡率。結(jié)果多重PCR體系檢測(cè)10種目標(biāo)細(xì)菌的標(biāo)準(zhǔn)菌株均呈現(xiàn)陽(yáng)性結(jié)果。濃度梯度敏感性實(shí)驗(yàn)提示本項(xiàng)目設(shè)計(jì)的多重PCR反應(yīng)體系的最小可檢測(cè)細(xì)菌濃度為1×103cfu/ml,最適宜檢測(cè)濃度范圍1×103cfu/ml～1×106cfu/ml。多重PCR組檢出率(81.8%)顯著高于對(duì)照組(10%),兩種檢驗(yàn)方法陽(yáng)性率差異有統(tǒng)計(jì)學(xué)意義(P0.05)。多重PCR組患者SIRS持續(xù)時(shí)間、抗生素費(fèi)用與對(duì)照組的差異有統(tǒng)計(jì)學(xué)意義(P0.05)。兩組患者住院總時(shí)間、抗菌藥物使用時(shí)間、住院總費(fèi)用及并發(fā)癥的發(fā)生率和死亡率的差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論本項(xiàng)目建立的多重PCR反應(yīng)體系具備高通量、精準(zhǔn)檢測(cè)膿毒癥患者感染的常見(jiàn)病原菌的能力。初步的臨床研究提示該方法在指導(dǎo)腸源性膿毒癥患者的抗微生物治療中有一定的應(yīng)用價(jià)值。
[Abstract]:Construction and preliminary clinical application of multiplex PCR for detection of common pathogenic bacteria in enterogenic sepsis objective to construct a rapid and reliable multiplex PCR reaction system for detection of common pathogenic bacteria in enterogenic sepsis and to explore its clinical value. Methods primers and probes of complementary sequences were designed according to the 16sRNA specific fragments of 10 common pathogens of enterogenic sepsis (hereinafter referred to as target bacteria). The multiplex PCR reaction system was optimized and the specific test and concentration gradient sensitivity test were carried out. 42 patients with enterogenic sepsis were randomly divided into multiple PCR treatment group (multiple PCR group) and empirical drug use group and blood culture guidance group (control group). The use time of antibiotics, the total cost of antibiotics, the total hospitalization cost, the duration of SIRS and the incidence and mortality of various complications were observed. Results the standard strains of 10 target bacteria detected by multiplex PCR system showed positive results. The concentration gradient sensitivity experiment indicates that the minimum detectable bacterial concentration of the multiplex PCR reaction system designed in this project is 1 脳 10 ~ 3 CFU / ml, and the most suitable detection concentration range is 1 脳 103cfu/ml~1 脳 10 ~ 6 CFU / ml. The positive rate of multiplex PCR group (81.8%) was significantly higher than that of control group (10%). There was significant difference between the two methods (P 0.05). The duration of SIRS and the cost of antibiotics in multiple PCR group were significantly different from those in control group (P 0.05). There was no significant difference in the total hospitalization time, the use time of antibiotics, the total cost of hospitalization and the incidence and mortality of complications between the two groups (P 0.05). Conclusion the multiplex PCR reaction system established in this project has the ability of high throughput and accurate detection of common pathogens in sepsis patients. Preliminary clinical studies suggest that this method has certain application value in guiding antimicrobial therapy in patients with enterogenic sepsis.
【學(xué)位授予單位】：浙江中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R459.7

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