建立并应用实时聚合酶链反应方法检测空肠弯曲菌。
Development and application of a real-time polymerase chain reaction method for Campylobacter jejuni detection.
机构信息
State Key Laboratory for Infectious Disease Prevention and Control, and National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
出版信息
World J Gastroenterol. 2013 May 28;19(20):3090-5. doi: 10.3748/wjg.v19.i20.3090.
AIM
To develop a real-time polymerase chain reaction (PCR) method to detect and quantify Campylobacter jejuni (C. jejuni) from stool specimens.
METHODS
Primers and a probe for real-time PCR were designed based on the specific DNA sequence of the hipO gene in C. jejuni. The specificity of the primers and probe were tested against a set of Campylobacter spp. and other enteric pathogens. The optimal PCR conditions were determined by testing a series of conditions with standard a C. jejuni template. The detection limits were obtained using purified DNA from bacterial culture and extracted DNA from the stool specimen. Two hundred and forty-two specimens were analyzed for the presence of C. jejuni by direct bacterial culture and real-time PCR.
RESULTS
The optimal PCR system was determined using reference DNA templates, 1 × uracil-DNA glycosylase, 3.5 mmol/L MgCl2, 1.25 U platinum Taq polymerase, 0.4 mmol/L PCR nucleotide mix, 0.48 μmol/L of each primer, 0.2 μmol/L of probe and 2 μL of DNA template in a final volume of 25 μL. The PCR reaction was carried as follows: 95 °C for 4 min, followed by 45 cycles of 10 s at 95 °C and 30 s at 59 °C. The detection limit was 4.3 CFU/mL using purified DNA from bacterial culture and 10(3) CFU/g using DNA from stool specimens. Twenty (8.3%, 20/242) C. jejuni strains were isolated from bacterial culture, while 41 (16.9%, 41/242) samples were found to be positive by real-time PCR. DNA sequencing of the PCR product indicated the presence of C. jejuni in the specimen. One mixed infection of C. jejuni and Salmonella was detected in one specimen and the PCR test for this specimen was positive.
CONCLUSION
The sensitivity of detection of C. jejuni from stool specimens was much higher using this PCR assay than using the direct culture method.
目的
开发一种实时聚合酶链反应（PCR）方法，用于检测和定量粪便标本中的空肠弯曲菌（C. jejuni）。
方法
根据空肠弯曲菌 hipO 基因的特异性 DNA 序列，设计了实时 PCR 的引物和探针。通过对一组弯曲菌属和其他肠道病原体的检测，测试了引物和探针的特异性。通过用标准 C. jejuni 模板测试一系列条件，确定了最佳 PCR 条件。使用从细菌培养物中提取的纯化 DNA 和从粪便标本中提取的 DNA 获得检测限。通过直接细菌培养和实时 PCR 分析 242 份标本中 C. jejuni 的存在情况。
结果
使用参考 DNA 模板确定了最佳 PCR 系统，该系统使用 1×尿嘧啶-DNA 糖基化酶、3.5mmol/L MgCl2、1.25U 白金 Taq 聚合酶、0.4mmol/L PCR 核苷酸混合物、0.48μmol/L 每种引物、0.2μmol/L 探针和 2μL DNA 模板，最终体积为 25μL。PCR 反应如下：95℃ 4 分钟，然后进行 45 个循环，95℃ 10 秒，59℃ 30 秒。使用从细菌培养物中提取的纯化 DNA 的检测限为 4.3CFU/mL，使用从粪便标本中提取的 DNA 的检测限为 10（3）CFU/g。从细菌培养物中分离出 20（8.3%，20/242）株空肠弯曲菌，而 41（16.9%，41/242）份标本通过实时 PCR 检测为阳性。PCR 产物的 DNA 测序表明标本中存在空肠弯曲菌。一份标本中检测到空肠弯曲菌和沙门氏菌的混合感染，该标本的 PCR 检测为阳性。
结论
与直接培养法相比，该 PCR 检测法检测粪便标本中空肠弯曲菌的灵敏度要高得多。
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