title: 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.
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.
  
  
  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.
authors.affiliation: Laboratory Medical Centre, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China; Clinical Laboratory, Bao'an Maternity and Child Health Hospital, Shenzhen, Guangdong Province, China.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Wu
authors.affiliation: Clinical Laboratory, The Fifth Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong Province, China.
authors.forename: Xiaomao
authors.initials: X
authors.lastname: Yin
authors.affiliation: Laboratory Medical Centre, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Zheng
authors.affiliation: Clinical Laboratory, Bao'an Maternity and Child Health Hospital, Shenzhen, Guangdong Province, China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Zou
authors.affiliation: Clinical Laboratory, Bao'an Maternity and Child Health Hospital, Shenzhen, Guangdong Province, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Jin
authors.affiliation: Laboratory Medical Centre, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China.
authors.forename: Yanwei
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Charles Sturt University, Leeds Parade, Orange, New South Wales, Australia.
authors.forename: Timothy
authors.initials: T
authors.lastname: Kudinha
authors.affiliation: Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research-Pathology West, Westmead Hospital, New South Wales, Australia.
authors.forename: Fanrong
authors.initials: F
authors.lastname: Kong
authors.affiliation: Clinical Laboratory, Bao'an Maternity and Child Health Hospital, Shenzhen, Guangdong Province, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Chen
authors.affiliation: Laboratory Medical Centre, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
mesh_terms: D002648:Child; D002681:China; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D006761:Hospitals; D006801:Humans; D058889:Molecular Typing / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009691:Nucleic Acid Denaturation; D011008:Pneumococcal Infections / Q000382:microbiology*; D015203:Reproducibility of Results; D012703:Serotyping / Q000379:methods*; D013296:Streptococcus pneumoniae / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D044366:Transition Temperature
chemical_list: D004269:DNA, Bacterial