Abstract
Background Streptococcus pneumoniae has more than 95 distinct serotypes described to date. However, only certain serotypes are more likely to cause pneumococcal diseases. Thus serotype surveillance is important for vaccine formula design as well as in post-vaccine serotype shift monitor. The goal of this study was to develop a practical screening assay for ten Shenzhen China common pneumococcal serotypes/serogroups in one molecular reaction.MethodsA molecular assay, based on multiplex ligation-dependent probe amplification (MLPA) and melting curve (MC) analysis, was developed in an integrated approach (MLPA-MC) for the detection of ten capsular serotypes/serogroups 4, 6 (6A/6B/6C/6D), 9V/9A, 14, 15F/15A, 15B/15C, 18 (18F/18A/18B/18C), 19F, 19A and 23F. We designed serotype/serogroup-specific MLPA probes and fluorescent detection probes to discriminate the different serotypes/serogroups in one molecular reaction. The three steps of MLPA-MC assay are continuous reactions in one well detected by LightCycler 480. A total of 210 S. pneumoniae isolates from our local Maternity and Child Health Hospital were randomly chosen to evaluate the assay against published multiplex PCR assays.ResultsOur results showed that 198 (94.3%) of S. pneumoniae isolates were type-able by our assays and the results were in complete concordance with the published multiplex PCRs. Using the MLPA-MC assay, 96 S. pneumoniae isolates could be typed within 3 hours with limited hands-on time. This serotype/serogroup-screening assay can be easily modified or extended by modification of the serotype/serogroup-specific MLPA probes combinations according to the needs of different laboratories.ConclusionsWe recommend use of this assay as a starting point for screening serotype/serogroup frequencies. There is a need for this assay to be combined with other molecular typing assays, like published serotype specific PCRs, or even the Quellung reaction for serotype confirmation.
Highlights
Streptococcus pneumoniae accounts for about 1 million children deaths annually due to pneumonia and meningitis, mostly in developing countries [1]
Our results showed that 198 (94.3%) of S. pneumoniae isolates were type-able by our assays and the results were in complete concordance with the published multiplex PCRs
Using the multiplex ligation-dependent probe amplification (MLPA)-melting curve (MC) assay, 96 S. pneumoniae isolates could be typed within 3 hours with limited hands-on time
Summary
Streptococcus pneumoniae accounts for about 1 million children deaths annually due to pneumonia and meningitis, mostly in developing countries [1]. The capsular polysaccharide represents an important virulence factor and characterizes S. pneumoniae into 95 distinct serotypes (within 46 serogroups). In China, two nation-wide studies on the distribution of S. pneumoniae serotypes in invasive pneumococcal diseases (IPD) and in children with pneumonia, showed that 19F, 19A, 23F, 6B and 14 were the most common serotypes, totally accounting for 73.1% of IPD and 87.9% of children with pneumonia [10, 11]. A study in Shenzhen city showed that these five serotypes (19F, 19A, 23F, 6B and 14), accounted for 81.6% of IPD in children[12]. Streptococcus pneumoniae has more than 95 distinct serotypes described to date. The goal of this study was to develop a practical screening assay for ten Shenzhen China common pneumococcal serotypes/serogroups in one molecular reaction
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