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首页> 外文期刊>Journal of Clinical Microbiology >Novel Pan-Genomic Analysis Approach in Target Selection for Multiplex PCR Identification and Detection of Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia cepacia Complex Species: a Proof-of-Concept Study
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Novel Pan-Genomic Analysis Approach in Target Selection for Multiplex PCR Identification and Detection of Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia cepacia Complex Species: a Proof-of-Concept Study

机译：靶标选择中的新型全基因组分析方法，用于多重PCR鉴定和检测伯克霍尔德氏菌，泰国伯克霍尔德氏菌和洋葱伯克霍尔德氏菌复杂物种：概念验证研究

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Burkholderia pseudomallei, Burkholderia thailandensis, and the Burkholderia cepacia complex differ greatly in pathogenicity and epidemiology. Yet, they are occasionally misidentified by biochemical profiling, and even 16S rRNA gene sequencing may not offer adequate discrimination between certain species groups. Using the 23 B. pseudomallei, four B. thailandensis, and 16 B. cepacia complex genome sequences available, we identified gene targets specific to each of them (a Tat domain protein, a 70-kDa protein, and a 12-kDa protein for B. pseudomallei, B. thailandensis, and the B. cepacia complex, respectively), with an in-house developed algorithm. Using these targets, we designed a robust multiplex PCR assay useful for their identification and detection from soil and simulated sputum samples. For all 43 B. pseudomallei, seven B. thailandensis, and 20 B. cepacia complex (B. multivorans, n = 6; B. cenocepacia, n = 3; B. cepacia, n = 4; B. arboris, n = 2; B. contaminans, B. anthina, and B. pyrrocinia, n = 1 each; other unnamed members, n = 2) isolates, the assay produced specific products of predicted size without false positives or negatives. Of the 60 soil samples screened, 19 (31.6%) and 29 (48.3%) were positive for B. pseudomallei and the B. cepacia complex, respectively, and in four (6.7%) soil samples, the organisms were codetected. DNA sequencing confirmed that all PCR products originated from their targeted loci. This novel pan-genomic analysis approach in target selection is simple, computationally efficient, and potentially applicable to any species that harbors species-specific genes. A multiplex PCR assay for rapid and accurate identification and detection of B. pseudomallei, B. thailandensis, and the B. cepacia complex was developed and verified.
机译：假伯克霍尔德氏菌，泰国伯克霍尔德氏菌和洋葱伯克霍尔德氏菌复合体的致病性和流行病学差异很大。然而，它们有时会因生化分析而被误认，甚至16S rRNA基因测序也可能无法对某些物种进行充分区分。使用可用的23 B. pseudomallei，4 B. thailandensis和16 B. cepacia复合基因组序列，我们鉴定了特异于它们的基因靶标（Tat域蛋白，70 kDa蛋白和12 kDa蛋白， B. pseudomallei，B。thailandensis和B. cepacia复合体），采用内部开发的算法。利用这些靶标，我们设计了一种强大的多重PCR分析方法，可用于从土壤和模拟痰液样品中进行鉴定和检测。对于所有43个拟假芽孢杆菌，7个泰国芽孢杆菌和20个洋葱曲霉菌复合体（B. multivorans， n = 6; B. cenocepacia， n = 3; B 。cepacia， n = 4; B。arboris， n = 2; B。contaminans，B。anthina和B. pyrrocinia， n = 1;其他未命名的成员， n = 2）分离物，该测定产生了预期大小的特定产物，没有假阳性或阴性。在筛选出的60个土壤样品中，分别有19个（31.6％）和29个（48.3％）的假苹果芽孢杆菌和洋葱伯克霍尔德菌复合体呈阳性，在四个（6.7％）土壤样品中，对这些生物进行了编码。 DNA测序证实所有PCR产物均来自其靶基因座。这种用于目标选择的新颖的全基因组分析方法简单，计算效率高，并且可能适用于任何具有物种特异性基因的物种。开发并验证了用于快速准确鉴定和检测假小芽孢杆菌，泰国芽孢杆菌和洋葱伯克霍尔德氏菌复合物的多重PCR检测方法。

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