Molecular Typing of ccrB Gene in Methicillin-resistant Staphylococcus aureus by Restriction Fragment Length Polymorphism

A typing procedure for Staphylococcus aureus was developed based on improved PCR amplification of the coagulase gene and restriction fragment length polymorphism (RFLP) analysis of the product. All coagulase-positive staphylococci produced a single PCR amplification product of either 875, 660, 603, or 547 bp. Those strains of epidemic methicillin-resistant S. aureus 16 (EMRSA-16) studied all gave a product of 547 bp. PCR products were digested withAluI and CfoI, and the fragments were separated by gel electrophoresis. Ten distinct RFLP patterns were found among 85 isolates of methicillin-resistant S. aureus (MRSA) and 10 propagating strains (PS) of methicillin-sensitive S. aureus(MSSA) examined. RFLP patterns 1, 2, and 3 were specific to strains of EMRSA-3, -15, and -16, respectively. By contrast, RFLP patterns 4 and 5 were seen with a heterogeneous collection of strains, together with drug-resistant forms of S. aureus isolated in Europe and four propagating strains used for the international phage set. RFLP pattern 6 was given by the Airedale isolate and PS 95. RFLP pattern 7 encompassed EMRSA-2 (isolate 331), PS 94, and PS 96. An isolate from Germany gave RFLP pattern 8. Eight strains of MSSA gave patterns similar to those of methicillin-resistant strains (RFLP patterns 3, 4, 5, 6, and 7), but two, PS 42E and PS 71, gave unique RFLP patterns 9 and 10, respectively. The coagulase gene PCR products for 24 isolates of MRSA and two isolates of MSSA were sequenced for both strands. The sequences were aligned, and evolutionary lineages were inferred based on pairwise distances between isolates.

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Human Lymphocyte Antigen Molecular Typing

Archives of Pathology & Laboratory Medicine ◽

10.5858/2002-126-0281-hlamt ◽

2002 ◽

Vol 126 (3) ◽

pp. 281-284

Author(s):

John A. Gerlach

Keyword(s):

Restriction Fragment Length Polymorphism ◽

Restriction Fragment ◽

Length Polymorphism ◽

Human Lymphocyte ◽

Fragment Length ◽

Molecular Typing ◽

Fragment Length Polymorphism ◽

Hla Typing ◽

Lymphocyte Antigen ◽

Restriction Fragment Length

Abstract The human lymphocyte antigen (HLA) typing community was one of the early groups to adopt molecular testing. This action was borne out of the need to identify the many alleles of the highly polymorphic HLA system. Early paradigms used restriction fragment length polymorphism regimes, but the polymerase chain reaction method of amplification quickly replaced that less-than-discriminating choice. Methods currently in use for HLA typing, with commercial kits available, are sequence-specific oligonucleotide probe (both dot blot and the reverse blot dot), sequence-specific primer amplification, restriction fragment length polymorphism of amplified products, double-stranded sequence conformation polymorphism (with and without reference strand), sequence-based typing, and microarray technologies. More than 1250 alleles are recognized by the World Health Organization and meet their criteria for assignment. These alleles can be identified by molecular methods and represent alleles present at class I and class II loci of the HLA complex. On occasion, ambiguous results still persist, even with the best molecular typing methods. Therefore, it is clear to the HLA typing community that a combination of the above methods may be needed to allow true discrimination of the possible alleles an individual carries in their genetic makeup. It is also clear that a typing laboratory may need to resort to nonmolecular serology to understand the significance and impact of the type generated by the HLA molecular typing laboratory.

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