Practical Prediction of Ten Common Streptococcus pneumoniae Serotypes/Serogroups in One PCR Reaction by Multiplex Ligation-Dependent Probe Amplification and Melting Curve (MLPA-MC) Assay in Shenzhen, China

PLOS ONE, Dec 2019

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. Methods A 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. Results 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 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. Conclusions We 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.

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Practical Prediction of Ten Common Streptococcus pneumoniae Serotypes/Serogroups in One PCR Reaction by Multiplex Ligation-Dependent Probe Amplification and Melting Curve (MLPA-MC) Assay in Shenzhen, China

July Practical Prediction of Ten Common Streptococcus pneumoniae Serotypes/ Serogroups in One PCR Reaction by Multiplex Ligation-Dependent Probe Amplification and Melting Curve (MLPA-MC) Assay in Shenzhen, China Lijuan Wu 0 1 Xiaomao Yin 0 1 Lei Zheng 0 1 Jianhua Zou 0 1 Ping Jin 0 1 Yanwei Hu 0 1 Timothy Kudinha 0 1 Fanrong Kong 0 1 Xu Chen 0 1 Qian Wang 0 1 Paulo Lee Ho, Instituto Butantan, BRAZIL 0 1 Laboratory Medical Centre, Nanfang Hospital, Southern Medical University , Guangzhou, Guangdong Province , China , 2 Clinical Laboratory, Bao'an Maternity and Child Health Hospital , Shenzhen, Guangdong Province, China, 3 Clinical Laboratory , The Fifth Affiliated Hospital of Southern Medical University , Guangzhou, Guangdong Province , China , 4 Charles Sturt University , Leeds Parade, Orange, New South Wales , Australia , 5 Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research-Pathology West, Westmead Hospital , New South Wales , Australia 1 Funding: This work was supported by the grant of Science and Technology plan of Bureau of Health of Shenzhen Municipality; China (201302168) to LW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript China - Competing Interests: The authors have declared that no competing interests exist. 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. A 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/serogroupspecific 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. 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 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. We 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. 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). There are ten common serotypes/serogroups that account for most paediatric pneumococcal infections worldwide, with serogroups 6, 14, 19 and 23, the most common [2–5]. However, the distribution of serotypes can vary with age, geography and time [2, 6–9]. 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]. The Quellung reaction or Neufeld test (conventional serotyping) is the gold standard for serotyping, but it’s too expensive for most developing countries including China [9, 10, 13, 14]. In addition, it’s labor-intensive and the interpretation of results is subjective and need expert training. Several different molecular assays have been developed as alternatives to serotyping, with most assays based on serotype-specific or serotype-associated sequences in the cps gene cluster, namely wzy and wzx, or cpsA-cpsB (wzg-wzh) [9, 15–17]. USA CDC have developed and published a sequential multiplex PCR scheme based on serotype specific PCRs to detect specific pneumococcal serotypes from isolates and (...truncated)


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Lijuan Wu, Xiaomao Yin, Lei Zheng, Jianhua Zou, Ping Jin, Yanwei Hu, Timothy Kudinha, Fanrong Kong, Xu Chen, Qian Wang. Practical Prediction of Ten Common Streptococcus pneumoniae Serotypes/Serogroups in One PCR Reaction by Multiplex Ligation-Dependent Probe Amplification and Melting Curve (MLPA-MC) Assay in Shenzhen, China, PLOS ONE, 2015, Volume 10, Issue 7, DOI: 10.1371/journal.pone.0130664