Identification of Pneumococcal Serotypes by PCR–Restriction Fragment Length Polymorphism

Abstract

Streptococcus pneumoniae shows more than 90 capsular serotypes that can be distinguished by their reactivity against antisera. The main objective of this work was the development of a molecular method for serotyping without the use of antisera. A computer program containing an algorithm was used to search in a database for potentially useful enzymes for Restriction Fragment Length Polymorphism-RFLP typing, in order to maximize the discrimination between different serotypes. DNA sequences of 90 serotypes for the region between dexB and aliA genes were compiled, and a computer screening of restriction enzymes was performed. The wzg–wzh–wzd–wze region and Sse9I restriction predicted unique PCR-RFLP patterns for 39 serotypes and eight serogroups. A second restriction enzyme resolved fragment specific patterns for 25 serotypes. The method was tested with 98 serotype-unknown clinical isolates. PCR-RFLP analysis deduced correct serotypes that were confirmed by Quellung reaction for 78.5% of the isolates.

The Molecular Approaches and Challenges of Streptococcus pneumoniae Serotyping for Epidemiological Surveillance in the Vaccine Era

“…PCR-based serotyping approaches like PCR-RFLP, multiplex PCR, real-time PCR, and sequetyping were devised as substitutes to agglutination approach for amplifying specific capsular DNA elements to offer an economical, sensitive, and relatively straightforward serotyping than conventional methods ( Lawrence et al 2003 ; Leung et al 2012 ; García-Suárez et al 2019 ). Employing a PCR-specific reaction in serotyping is advantageous because it facilitates serotype detection, specifically when conventional techniques are challenged with the cost of antisera and constraints in getting good bacterial culture due to the fastidious nature of certain serotypes ( Gonzales-Siles et al 2019 ).…”

“… Botstein et al (1980) first described RFLP that produced a genetic association map for humans; it was the first DNA profiling approach used extensively for gene description and genome mapping ( Botstein et al 1980 ; Dai and Long 2015 ). The PCR-RFLP approach is based on the amplification of a particular DNA target areas and digestion based on restriction enzymes to produce several distinctly-sized DNA fragments to enable distinguishing between distinct serotypes ( García-Suárez et al 2019 ; Lawrence et al 2000 ). Restriction enzymes used in the PCR-RFLP technique make use of the cps polymorphisms in pneumococci for serotype detection ( Camargo et al 2015 ).…”

“…Numerous studies employed the PCR-RFLP technique; however, the cpsA - cpsB and dexB - aliA regions contain extensive fragments, requiring the selection of a sequence corresponding to the cps gene and the specific restriction enzyme set for generating fragments ( García-Suárez et al 2019 ). Moreover, PCR amplification of a regulatory area between the cpsA and cpsB locus based on three distinct restriction enzymes ( AluI, HinfI , and RsaI ) facilitates distinguishing the serogroup level, while the specific serotype cannot be distinguished ( Lawrence et al 2000 ).…”

“…Hence, the XhoII -augmented cps-RFLP technique produced a superior outcome than antisera and other molecular serotyping approaches. The most recent PCR-RFLP version specifies a serotyping approach based on the same PCR primers for amplifying comparatively minor regulatory areas between cpsA - cpsD and an RFLP process using Sse9I for resolving serogroup and serotype ( García-Suárez et al 2019 ). Sse9I digestion-based cps analysis offered a potent tool for precise serotyping of the most prevalent S. pneumoniae serotypes (1, 3, 6A, 14, and 19A, including non-PCV13 serotypes such as 24F, 23B, and 10A) ( García-Suárez et al 2019 ).…”