South African Medical Research Council, Vaccines and Infectious Diseases Analytics Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.
Department of Science and Technology/National Research Foundation, South African Research Chair Initiative in Vaccine Preventable Diseases, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.
Sci Rep. 2021 Dec 9;11(1):23728. doi: 10.1038/s41598-021-03127-9.
Current real-time high-throughput Polymerase Chain Reaction (qPCR) methods do not distinguish serotypes 6A from 6B, 18C from 18A/B and 22F from 22A. We established a nanofluidic real-time PCR (Fluidigm) for serotyping that included Dual-Priming-Oligonucleotides (DPO), a Locked-Nucleic-Acid (LNA) probe and TaqMan assay-sets for high-throughput serotyping. The designed assay-sets target capsular gene wciP in serogroup 6, wciX and wxcM in serogroup 18, and wcwA in serogroup 22. An algorithm combining results from published assay-sets (6A/B/C/D; 6C/D; 18A/B/C; 22A/F) and designed assay-sets for 6A/C; 18B/C/F; 18C/F, 18F and 22F was validated through blind analysis of 1973 archived clinical samples collected from South African children ≤ 5-years-old (2009-2011), previously serotyped with the culture-based Quellung method. All assay-sets were efficient (92-101%), had low variation between replicates (R > 0.98), and were able to detect targets at a limit of detection (LOD) of < 100 Colony-Forming-Units (CFU)/mL of sample. There was high concordance (Kappa = 0.73-0.92); sensitivity (85-100%) and specificity (96-100%) for Fluidigm compared with Quellung for serotyping 6A; 6B; 6C; 18C and 22F. Fluidigm distinguishes vaccine-serotypes 6A, 6B, 18C, next-generation PCV-serotype 22F and non-vaccine-serotypes 6C, 6D, 18A, 18B, 18F and 22A. Discriminating single serotypes is important for assessing serotype replacement and the impact of PCVs on vaccine- and non-vaccine serotypes.
目前的实时高通量聚合酶链反应（qPCR）方法无法区分血清型 6A 与 6B、18C 与 18A/B 和 22F 与 22A。我们建立了一种用于血清分型的纳米流控实时 PCR（Fluidigm），包括双引物寡核苷酸（DPO）、锁定核酸（LNA）探针和用于高通量血清分型的 TaqMan 检测试剂盒。设计的检测试剂盒针对血清群 6 的荚膜基因 wciP、血清群 18 的 wciX 和 wxcM 以及血清群 22 的 wcwA。一种算法结合了已发表的检测试剂盒（6A/B/C/D；6C/D；18A/B/C；22A/F）和用于 6A/C；18B/C/F；18C/F、18F 和 22F 的设计检测试剂盒的结果，通过对 1973 份来自南非 5 岁以下儿童（2009-2011 年）的存档临床样本进行盲法分析进行验证，这些样本之前曾用基于培养的 Quellung 方法进行过血清型鉴定。所有检测试剂盒均高效（92-101%），重复间变异小（R>0.98），能够检测到样本中低至 100 个集落形成单位（CFU）/mL 的目标。Fluidigm 与 Quellung 相比，在血清型 6A；6B；6C；18C 和 22F 的一致性（Kappa=0.73-0.92）、敏感性（85-100%）和特异性（96-100%）均较高。Fluidigm 可区分疫苗血清型 6A、6B、18C、新一代 PCV 血清型 22F 和非疫苗血清型 6C、6D、18A、18B、18F 和 22A。区分单一血清型对于评估血清型替代以及 PCV 对疫苗和非疫苗血清型的影响非常重要。
