种特异性PCR快速检测奶粉中阪崎肠杆菌研究

Rapid Detection for Enterobacter sakazakii in Powdered Milks Based on Species-specific PCR

摘要:

阪崎肠杆菌是一种目前认为以奶粉为传播媒介的食源性条件致病菌，通过α-1,4-葡萄糖苷酶基因和ompA基因分别设计引物ESF-ESR和ESSF-ESSR，进行单重和双重PCR方法研究，结果显示所有阪崎肠杆菌菌株PCR扩增均为阳性，阴性对照均未扩增出目的片段；纯菌单重PCR灵敏度分别为10²cfu/mL和10¹cfu/mL，双重PCR灵敏度为10³cfu/mL；在有或无其他细菌存在时，人工污染阪崎肠杆菌模拟样品单重PCR检测灵敏度分别为10³cfu/mL和10²cfu/mL，双重PCR检测灵敏度为10⁴cfu/mL；实际样品检测显示PCR方法与传统方法具有很好的一致性。结果表明：该PCR方法具有很好的种特异性和灵敏度，能够克服奶粉中杂菌对快速检测阪崎肠杆菌造成的干扰，减少以保守序列来设计引物导致假阳性结果的出现，可以较好地应用于奶粉中阪崎肠杆菌的检测与鉴定。

Abstract:

Enterobacter sakazakii was an emerging food-borne pathogen associated with meningitis, sepsis and necrotizing enterocolitis, especially in neonates with high potential danger. The conventional detection methods were time-consuming and operated arduously, sometimes gave ambiguous signals. Increasing of reports showed the infant formula was the main infection vehicle. Consequently, it was important that improvement of methods for earlier detection and confirmation of presumptive E.sakazakii in infant formula to control and guard against the epidemic diseases due to E.sakazakii. In this study, PCR assay was developed based on ompA and α-1, 4-glusidase genes. Expected fragments(469 bp and 673 bp)were produced from 8 strains of E. sakazakii including E.sakazakii ATCC51329 after PCR amplification, but not from 70 strains of other bacteria. The sensitivity is 10¹ cfu/mL and 10² cfu/mL by signal-PCR using ESSF-ESSR and ESF-ESR as primers respectively in pure culture, sensitivity of dual-PCR is 10³ cfu/mL. Detection limit in artificially contaminated infant formula is 10² cfu/mL and 10³ cfu/mL by single-PCR with ESF-ESR and ESSF-ESSR respectively. To investigate whether the presence of other bacteria in infant formula have any effect on the sensitivity of PCR, two sets of experiment were designed. Different levels of other bacterial do not affect the PCR detection limit, which indicates the primers are species-specific for E.sakazakii. The investigation of actual samples shows that PCR assay is consistent with FDA standard method. The new method developed in the study can be used widely to detect the presence of E.sakazakii in infant formula with higher sensitivity and specificity than conventional methods.