Molecular Typing of Staphylococcus aureus Based on PCR Restriction Fragment Length Polymorphism and DNA Sequence Analysis of the Coagulase Gene

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 with AluI 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.     

Restriction fragment length polymorphism analysis of open reading frame 5 gene of porcine reproductive and respiratory syndrome virus isolates in Korea

Summary.  The genetic variability of porcine reproductive and respiratory syndrome virus (PRRSV) was studied by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)-amplified fragments among 50 Korean isolates from open reading frame 5. All Korean PRRSVs were isolated from the field cases after the marketing of an U.S. ATCC VR2332-derived modified live PRRSV vaccine. Combining the restriction enzyme digestion patterns obtained with MluI, HincII, SacII, and HaeIII, we observed 19 distinct RFLP patterns. Seventeen out of 50 PRRSV isolates (34%) exhibited the modified live PRRSV vaccine RFLP pattern. The genomic variations that have been identified in the present study seemed to represent characteristic features of the Korean PRRSV isolates. PCR-based RFLP analysis using several restriction enzymes provides a good genetic estimate for isolate differentiation. Received December 20, 1999 Accepted January 28, 2000     

Homogeneity and diversity of genome polymorphism in a set of herpes simplex virus type 1 strains classified as the same genotypic group

Summary Restriction fragment length polymorphism (RFLP) of 327 strains of herpes simplex virus type 1 (HSV-1) isolated in Japan were analyzed using six restriction endonucleases (REs) recognizing 6 base pairs (BamHI,HindIII,KpnI,PvuII,SalI,XhoI). The presence of strains with the same RFLP profile became evident. Fifteen strains of each of the two predominant sets with the same RFLP profile were further analyzed using two different methods, that is analyses of RFLP using 3 restriction endonucleases recognizing 4 base pairs (4-bp REs) (HaeIII,HhaI,MboI) and analyses of the variation of 3 reiterated sequences (reiterations I, IV, VII). Most of the epidemiologically unrelated strains could be differentiated by variation of the reiterations. RFLPs differentiating the strains were detected with the 4-bp REs, and epidemiologically related strains shared a specific RFLP, thereby confirming the relationship. These results verified the utility of the two methods for use in molecular epidemiological studies. Homogeneity of RFLP among the strains suggested derivation from a common ancestor (making up a genotypic group) while diversity in strains of the same genotypic group was indicative of a separate replication.     

Genetic characterization of E2 gene of classical swine fever virus by restriction fragment length polymorphism and phylogenetic analysis

An RT-nested PCR (RT-nPCR)-based restriction fragment length polymorphism (RFLP) analyses of the E2 gene were developed for genetic subtyping and differentiation of vaccinated and infected classical swine fever virus (CSFV) strains. RT-nPCR identified 96 CSFV-positive samples from 321 clinical specimens from southeastern China during 2003–2008. The PCR products of positive samples were further differentiated using MspI digestion, 23 were identified as the C-strain, 62 as field strains, and 11 as mixture of the vaccine strain and field ones. RFLP with BglI, DdeI, DraI, and PstI were then used for subtyping of the field CSFV isolates. Thirty-eight field isolates phylogenetically classified as subgroup 2.1 based on E2 were divided into 11 subtypes by this RFLP scheme. Both RFLP profiling and sequence-based phylogenetic analysis revealed genetic diversity of CSFV in the field. Three novel substitutions at amino acid positions 17, 93, and 286 were identified in the predominant subtype VI strains isolated in 2008 as compared to other strains including historical subtype VI strains. These results suggest that CSFV in China experienced gradual variations and evolutionary accumulation progress. Thus, the RFLP methods targeting on the CSFV E2 gene are suitable for epidemiological survey in endemic area where the C-strain is applied for vaccination. Combination of the RFLP schemes with sequence-based phylogenetic analysis could provide more detailed information on transmission of CSFV in the region or even its evolution.     

Restriction fragment length polymorphism of PCR amplifiedpapE gene products is correlated with complete serotype among uropathogenicEscherichia coliisolates

Using whole bacteria, rather than extracted, purified DNA samples, we amplified thepapE gene sequences from 63Escherichia coliisolates belonging to O serogroups O1, O2 and O6. These isolates were from collections separated temporally as well as geographically: from four cities in the US and one in Sweden. PCR amplifiedpapE products were digested with restriction endonucleases and the relative sizes of the fragments compared for each strain. For 41 of the strains, we found a correlation between thepapE restriction fragment length polymorphism (RFLP) and the complete serotype. Furthermore, we were able to detect the presence of duplicate copies of the gene in 14 of the isolates; these 14 isolates were among the 22 that did not exhibit a correlation between the RFLP of their amplifiedpapE sequences and their complete serotype. We conclude that RFLP analysis of PCR products is a rapid and relatively simple method for examining the DNA ofE. colicontaining thepapgene sequence.     

A new perspective on and re-assessment of SAG2 locus as the tool for genetic analysis of <Emphasis Type="BoldItalic">Toxoplasma gondii</Emphasis> isolates

SAG2 locus, the coding gene of the P22 protein, has been widely used for the molecular epidemiology of Toxoplasma gondii and characterization of the parasite isolates with two separate polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) processes. To re-assess the resolution power and suitability of this genetic marker for molecular characterization of the parasite isolates, a number of 27 Toxoplasma strains from different zymodeme patterns were used in the present study. Both codon and non-codon regions of the SAG2 locus of all 27 strains were amplified and subjected to sequencing and nucleotide alignment. Nucleotide variations clustered the three major genotypes (I, II and III). Some minor genotypes, unidentifiable by SAG2-RFLP, could be identified by sequence comparison. However, there were other genotypes that could not be differentiated from the major types due to having identical sequences. This suggests that a remarkable number of field isolates representing several minor types will be miss-clustered with the major types by using the traditional SAG2-PCR-RFLP method. It was concluded that this technique seems not to be suitable for Toxoplasma population study. Thus, the utilization of more variable markers and other discriminatory methods are also recommended.     

Multiplex PCR detection of vanA, vanB, vanC-1, and vanC-2/3 genes in enterococci.

Vancomycin-resistant enterococci (VRE) are increasingly isolated from clinical specimens. One hundred clinical isolates of enterococci (E. casseliflavus/E. flavescens [n = 10], E. faecalis [n = 34], E. faecium [n = 43], E. avium [n = 1], E. gallinarum [n = 11], and E. raffinosus [n = 1]) were examined for the presence of vanA, vanB, vanC-1, and vanC-2/3 genes by a single multiplex PCR performed directly with colonies from blood agar plates. Six previously characterized VRE strains which carry either vanA, vanB, vanC-1, or vanC-2 genes were used as controls. To discriminate among van genes, the PCR amplicons were digested with MspI and were electrophoresed on agarose gels. Because of significant sequence homology between vanC-2 and vanC-3 genes, this assay is unable to discriminate these genes from each other; therefore, these are referred to as vanC-2/3 genes. PCR products were detected in 63 of the 100 clinical isolates. The restriction fragment length patterns were consistent with vanA for 10 strains, vanB for 30 strains, vanC-1 for 12 strains, vanC-2 for 6 strains, and vanA and vanC-1 for 1 strain. The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanA and vanB were all > and = 64 micrograms/ml. The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanC-1 or vanC-2 were 4 to 8 micrograms/ml. The vancomycin MICs for the isolates from which no PCR amplicons were produced were 2 to 4 micrograms/ml. A PCR product was produced in four isolates (vancomycin MICs, 4 to > 256 micrograms/ml) with restriction fragment length patterns differing from those for the control vanA, vanB, vanC-1, and vanC-2 isolates. DNA sequencing of these amplicons revealed that two of the four isolates had nucleic acid sequences which were closely related to the published sequence for the vanB gene and two had nucleic acid sequences which were closely related to the published sequence for the vanC-2 and vanC-3 genes. Multiplex PCR-restriction fragment length polymorphism appears to be a useful and convenient method for rapidly detecting and discriminating genotypes for vancomycin-resistant Enterococcus spp. in the clinical laboratory. In instances in which unusual restriction fragment patterns of PCR amplicons occur, DNA sequencing can be performed to discriminate van genotypes.     

Typing of recent infectious bronchitis virus isolates causing nephritis in chicken

Summary Four isolates of infectious bronchitis viruses (IBV) from chickens with nephritis, were characterized by polymerase chain reaction (PCR) and restriction enzyme fragment length polymorphism (RFLP), and were found to be genetically different from the other twelve strains which we previously studied.     

Epidemiological studies on fowl adenoviruses isolated from cases of infectious hydropericardium

Serology, restriction enzyme analysis and polymerase chain reactions were used to classify a total of 12 fowl adenoviruses (FAV) isolated from clinical cases of infectious hydropericardium from field outbreaks in seven countries in Asia and America. All isolates belonged to FAV serotype 4. Two isolates were contaminated with avian adeno-associated virus and one of them also contained FAV1. Minor differences were observed in the BamHI restriction profiles. More variability was seen with SmaI, BglII and PstI restriction profiles. However, more than 80% of the fragments were identical in size in the five different PstI profiles, indicating the close genomic relationship between the isolates. Polymerase chain reaction assays supported the classification of the isolates as FAV4 strains. All isolates could be detected using H1/H2 or H3/H4 primer pairs. Restriction enzyme analysis of the H1/H2 polymerase chain reaction (PCR) products allowed no differentiation between the isolates, whereas the three isolates from India and Pakistan could be separated from all others after HpaII digestion of the H3/H4 PCR products. Although strain variation was demonstrated, it could be shown that all adenoviruses isolated from various field cases of infectious hydropericardium (Angara Disease) in several countries belong to fowl adenovirus serotype 4.     

Comparative Analysis of Haemophilus influenzae hifA (Pilin) Genes

Adherence of Haemophilus influenzae to epithelial cells plays a central role in colonization and is the first step in infection with this organism. Pili, which are large polymorphic surface proteins, have been shown to mediate the binding of H. influenzae to cells of the human respiratory tract. Earlier experiments have demonstrated that the major epitopes of H. influenzae pili are highly conformational and immunologically heterogenous; their subunit pilins are, however, immunologically homogenous. To define the extent of structural variation in pilins, which polymerize to form pili, the pilin genes (hifA) of 26 type a to f and 16 nontypeable strains of H. influenzae were amplified by PCR and subjected to restriction fragment length polymorphism (RFLP) analysis with AluI and RsaI. Six different RFLP patterns were identified. Four further RFLP patterns were identified from published hifA sequences from five nontypeable H. influenzae strains. Two patterns contained only nontypeable isolates; one of these contained H. influenzae biotype aegyptius strains F3031 and F3037. Another pattern contained predominantly H. influenzae type f strains. All other patterns were displayed by a variety of capsular and noncapsular types. Sequence analysis of selected hifA genes confirmed the 10 RFLP patterns and showed strong identity among representatives displaying the same RFLP patterns. In addition, the immunologic reactivity of pili with antipilus antisera correlated with the groupings of strains based on hifA RFLP patterns. Those strains that show greater reactivity with antiserum directed against H. influenzae type b strain M43 pili tend to fall into one RFLP pattern (pattern 3); while those strains that show equal or greater reactivity with antiserum directed against H. influenzae type b strain Eagan pili tend to fall in a different RFLP pattern (pattern 1). Sequence analysis of representative HifA pilins from typeable and nontypeable H. influenzae identified several highly conserved regions that play a role in bacterial pilus assembly and other regions with considerable amino acid heterogeneity. These regions of HifA amino acid sequence heterogeneity may explain the immunologic diversity seen in intact pili.     

Genotyping of Staphylococcus aureus isolated from bovine mastitis based on PCR-RFLP analysis of the aroA gene

The aim of this study was the genotypical characterization of 58 Staphylococcus aureus isolates from nine dairy herds in the Tabriz and Urmia regions of east and west Azerbaijan provinces, Iran. In this study, 58 S. aureus isolates from 370 milk samples from cows with clinical and subclinical mastitis were analyzed by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis of the aroA gene. Amplification of the aroA gene resulted in a single amplicon with a size of approximately 1,153 bp from all 58 isolates of S. aureus. To obtain the RFLP patterns of the isolates, the PCR products were digested with TaqI restriction enzyme and the fragments separated by gel electrophoresis. Four distinct RFLP patterns were observed among the studied isolates. Three out of four detected genotypes had the same RFLP patterns (A, B, and N) as reported by previous studies. The fourth newly detected genotype in this study was named H. Genotypes A and B were the most frequent, being observed in 24 (41.38%) and 29 (50%) isolates, respectively. Genotypes N and H comprised 1.7% and 6.9% of all isolates, respectively. With the exception of the RFLP pattern N, which was observed only in the Tabriz region, all other patterns were found in both Tabriz and Urmia regions. The results demonstrate that strain variations of S. aureus could occur within and between herds and also between different regions, although a few genotypes of S. aureus were predominant in bovine mastitis. This study also indicated that PCR amplification of the aroA gene is specific for S. aureus identification.     

Phenotypic and genotypic methods for the detection of herpes simplex virus serotypes

Typing of herpes simplex virus (HSV) into its serotypes plays a major role in epidemiology and management of reactivation. To develop and evaluate a polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) was employed using Hae III and Taq I against neutralization test, allele-specific PCR and DNA sequencing for the detection of HSV serotypes. Neutralization test, allele-specific PCR, DNA sequencing and PCR-based RFLP were applied simultaneously to 2 standard strains (HSV-1 and HSV-2) and 23 clinical isolates. PCR-based RFLP was applied further to 20 culture negative PCR positive clinical specimens. The 179 bp product of the clinical isolates and specimens amplified using the type-common primers of HSV was subjected to DNA sequencing and PCR-based RFLP. Allele-specific PCR was absolutely specific and highly sensitive. All the typing methods differentiated concordantly 23 clinical isolates into 12 HSV-1 and 11 HSV-2. DNA sequencing did not reveal any nucleotide variations within the serotypes among the isolates sequenced. PCR-based RFLP typed a further 20 culture negative clinical specimens into 15 HSV-1 and 5 HSV-2. PCR-based RFLP was a reliable, less laborious and cost-effective molecular biological tool for the determination of HSV serotypes both for the clinical isolates and culture negative specimens.     

Molecular subtyping of human T-lymphotropic virus type I (HTLV-I) by a nested polymerase chain reaction-restriction fragment length polymorphism analysis of the envelope gene: Two distinct lineages of HTLV-I in Taiwan

The major type of human T-lymphotropic virus type I (HTLV-I), generally referred to as the cosmopolitan type, has been grouped into three subtypes (A, B, and C) by phylogenetic analysis of the long terminal repeat sequences of the viral genome. Twelve subtype-specific nucleotide variations have been deduced by comparison between the envelope (env) sequences of 16 HTLV-I strains with defined subtypes and 9 Taiwanese HTLV-I strains. To gain further insights into the molecular epidemiology of HTLV-I and the origin of this virus in Taiwan, a rapid method of identification for the cosmopolitan subtypes was developed by using a nested polymerase chain reaction (PCR) and subsequent restriction fragment length polymorphism (RFLP) studies using the two subtype B-specific and four subtype C-specific nucleotides located within the positions 5708 to 6320 of the env gene. The nested PCR-RFLP method was used to subtype HTLV-I from four virus-positive cell lines derived from 1 Japanese and 3 North American patients, as well as 41 blood-unrelated Taiwan Chinese. The sequences of PCR products were determined and the six positions of subtype-specific nucleotide variations were examined. The sequence data completely supported the subtyping data via the nested PCR-RFLP method. The results demonstrated that, as is the case in Japan, at least two distinct cosmopolitan subtypes (A and B) of HTLV-I were present in Taiwan, but the more prevalent subtype in Taiwan is A in contrast to subtype B in Japan. Furthermore, rapid subtyping could facilitate molecular epidemiological studies of HTLV-I infection and linkage between HTLV-I subtypes and virus-associated diseases. J Med Virol 51:25–31, 1997. © 1997 Wiley-Liss Inc.     

Molecular Fingerprinting of Mycobacterium tuberculosis Isolates Obtained in Havana, Cuba, by IS6110 Restriction Fragment Length Polymorphism Analysis and by the Double-Repetitive-Element PCR Method

Mycobacterium tuberculosis sputum isolates from 38 patients, obtained in the first 6 months of 1997 in Havana, Cuba, were characterized by IS6110 restriction fragment length polymorphism (RFLP) analysis and the double-repetitive-element PCR (DRE-PCR) method. Among 41 strains from 38 patients, 24 and 25 unique patterns, and 5 and 4 cluster patterns, were found by the RFLP and DRE-PCR methods, respectively. Patients within two of these clusters were found to be epidemiologically related, while no relation was observed in patients in the other clusters. The DRE-PCR method is rapid, and it was as discriminating as IS6110 RFLP analysis in identifying an epidemiological association. Its simplicity makes the technique accessible for subtyping of M. tuberculosis strains in laboratories not equipped to perform RFLP analysis.     

Genetic variation in Staphylococcus aureus coagulase genes: potential and limits for use as epidemiological marker.

To perform coagulase gene typing, the repeated units encoding hypervariable regions of the Staphylococcus aureus coagulase gene were amplified by the PCR technique; this was followed by AluI restriction enzyme digestion and analysis of restriction fragment length polymorphism (RFLP) patterns. In order to assess the discriminatory power of this typing method, 30 epidemiologically unrelated S. aureus strains which differed by their pulsed-field gel electrophoresis patterns were examined. Although 18 of the 30 strains had unique and unshared AluI RFLP patterns, there were only four observed patterns in the remaining 12 strains. This finding indicated that unrelated strains may share identical AluI RFLP patterns. To elucidate the degree of genetic variation in the C-terminus-encoding loci within the coagulase genes, the PCR products of these 12 strains were subjected to Taq polymerase-mediated sequencing. Sequence analysis confirmed the AluI recognition sites in each of the four RFLP groups and demonstrated that AluI appears to yield the highest RFLP in restriction enzyme analysis. By their DNA sequences the majority of strains sharing common AluI groups could be clearly differentiated from each other and revealed between 93.2 and 98.5% homology. When we determined the nucleotide sequences of two strains after six subcultivations no significant alterations were observed. Because the discriminatory power of the current coagulase gene typing method is not great enough to be used as the sole method to type S. aureus, additional techniques are necessary. Sequence analysis of the repeated unit-encoding region for the typing of S. aureus may be potentially useful as an alternative to other current molecular typing techniques.