HLA分型方法的研究进展

通过对人类白细胞抗原(human leucocyte antigen,HLA)结构和功能的研究,有助于认识其在免疫学、人类学等领域中的作用,揭示其在疾病发生、发展中的作用机理,而对HLA分型研究却是认识其作用的关键一步。随着PCR技术的广泛应用,HLA基因分型方法得到了迅速的发展。本文对HLA基因分型技术的进展情况进行综述。     

Molecular typing methods for Neisseria meningitidis

Neisseria meningitidis is an important pathogen because it causes life-threatening infections. The rapid course of meningococcal disease and the capacity of some serogroups to cause large-scale epidemics necessitates the use of sensitive, reliable and rapid typing methods to characterise strains. Molecular typing techniques for N. meningitidis are used for epidemiological purposes to investigate outbreaks and the spread of organisms and to examine the population genetic structure of the organism to understand better its variation and evolution. Many investigators have employed molecular typing methods and shown that meningococcal disease is associated with a variety of different epidemiological patterns. The choice of a typing method is dependent upon the epidemiological questions to be answered and on the population genetics of the organism under investigation. With highly clonal populations comprising independent non-recombining lineages such as serogroup A meningococci, ribotyping, multilocus enzyme electrophoresis (MLEE), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), PCR with arbitrary primers (RAPD) or with other gene-based primers each provides a constant measure of the relationship between strains. A more restricted portfolio of molecular methods - PFGE, MLEE and MLST - is appropriate for the investigation of less clonal serogroup B and C meningococci from localised outbreaks. If a thorough evaluation of the overall population is sought to determine the relationship between new isolates and members of hyper-endemic clonal complexes then quantitative methods such as MLEE and MLST are necessary. Several PCR-based methods are used for the detection and typing of meningococcal strains, many requiring rigorous standardisation before they can be considered suitable for rapid and reliable differentiation between clones. This review examines strain characterisation by molecular techniques and non-culture-based subtyping of meningococci in clinical specimens. It assesses the importance of these techniques and examines the epidemiological questions that they answer and also their limitations.     

Evaluation of methods for typing coagulase-negative staphylococci.

One hundred and forty-two coagulase-negative staphylococci (CNS) isolated from dialysate effluent or skin of patients receiving continuous ambulatory peritoneal dialysis (CAPD) were typed by extended antibiogram (16 antibiotics) and biotype (26 reactions). These isolates were then typed by supplementary methods to determine the most suitable typing method for an epidemiological study of antibiotic resistance. These included phage typing, reverse phage typing, plasmid typing, whole-cell protein typing by SDS-PAGE with analysis by densitometry, and immunoblotting. The percentage of isolates typed successfully by the supplementary methods were: phage typing 20%, reverse phage typing 0%, plasmid typing 66%, SDS-PAGE 100%, immunoblotting 100%. The discrimination of each method was: phage typing 20%, plasmid typing 37%, SDS-PAGE 69%, immunoblotting 57%. Reproducibility was 88% for phage typing and 97% for plasmid typing. The reproducibility of the whole-cell protein typing was 83% if the same extracts were used but only 43% when separate protein extracts were analysed on separate occasions. However, strain relatedness was highly reproducible. The determination of an antibiogram-biotype profile was not a sufficiently accurate typing method for an epidemiological study of antibiotic resistance. Whole-cell protein typing by SDS-PAGE or immunoblotting was technically demanding but was the most effective of the supplementary methods for detecting erroneous discrimination and false matching produced by antibiogram-biotype combinations.     

Comparison of PCR-based methods for typing Escherichia coli

Objective  To establish a library typing system appropriate for studying cross-transmission of Escherichia coli.
Methods  Eighteen epidemiologically unrelated isolates were genotyped by means of pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD), repetitive (rep) PCR, and fluorescent amplified fragment length polymorphism (AFLP). Fingerprints were analyzed either by Pearson correlation or, in the case of AFLP, by Dice coefficients employing the novel 'uncertain band' software tool from GelCompar II. During a nine-month period, 112 isolates taken from 93 patients hospitalized in five intensive care units were analyzed by use of the two most discriminative PCR typing methods.
Results  Genotyping by RAPD and rep-PCR revealed insufficient discrimination. Among 18 epidemiologically unrelated strains with 17 different PFGE patterns, IS3 rep-PCR and AFLP distinguished 10 and 18 types, respectively. Comparison of the different methods for analysis of AFLP fingerprints showed that the Dice coefficients, which ignore 'uncertain bands', offered the best concordance with visual interpretation. Consecutive isolates originating from the same patient differed in less than three fragments.
Conclusions  AFLP analysis showed the highest discriminative capacity for PCR typing of E. coli isolates. Analysis of fingerprints employing the Dice coefficients proved the most efficient method for an automated software-based retrieval of visually indistinguishable genotypes in an AFLP fingerprint database.     

Comparison of three typing methods for Pseudomonas aeruginosa

Fifty-seven independent isolates of Pseudomonas aeruginosa from blood specimens were typed with 3 different methods: ribotyping, random amplified polymorphic DNA (RAPD) typing, and pyocin typing. Ribotyping was performed by probing the rRNA genes of genomic DNA that was digested separately with 4 different restriction enzymes. Digestion of DNA from 57 P. aeruginosa isolates with BamHI, ClaI, EcoRI, and PstI produced 4, 4, 6, and 7 patterns, respectively. As a result, ribotyping classified the 57 isolates into 22 types. Six new ribotypes that had not been described previously were found. One BamHI, 1 ClaI, 2 EcoRI, and 2 PstI patterns were novel. RAPD typing was performed with two different polymerase chain reaction (PCR) primers (RAPD1 and RAPD2). Both primers classified the 57 isolates into 15 RAPD types and produced identical patterns. The pyocin typing method classified the 57 isolates into 10 types. According to the results obtained in this study, the ribotyping has a discriminatory index of 0.865, RAPD, 0.785, and pyocin typing, 0.676, respectively. The ribotyping method was the most effective among the 3 methods compared for typing P. aeruginosa isolates.     

Comparison of multiple typing methods for Aspergillus fumigatus

As part of studies on the spread of infections, risk factors and prevention, several typing methods were developed to investigate the epidemiology of Aspergillus fumigatus . In the present study, 52 clinical isolates of A. fumigatus from 12 airway specimens from patients with invasive aspergillosis (hospitalized in three different centres) were characterized by short tandem repeat (STR) typing and multilocus sequence typing (MLST). These isolates were previously typed by random amplified polymorphic DNA (RAPD), sequence-specific DNA polymorphism (SSDP), microsatellite polymorphism (MSP) and multilocus enzyme electrophoresis (MLEE). STR typing identified 30 genotypes and, for most patients, all isolates were grouped in one cluster of the unweighted pair group method with arithmetic mean dendrogram. Using MLST, 16 genotypes were identified among 50 isolates, while two isolates appeared untypeable. RAPD, MSP, SSDP and MLEE allowed identification of eight, 14, nine and eight genotypes, respectively. Combining the results of these methods led to the delineation of 25 genotypes and a similar clustering pattern as with STR typing. In general, STR typing led to similar results to the previous combination of RAPD, SSDP, MSP and MLEE, but had a higher resolution, whereas MLST was less discriminatory and resulted in a totally different clustering pattern. Therefore, this study suggests the use of STR typing for research concerning the local epidemiology of A. fumigatus , which requires a high discriminatory power.     

DNA-RFLP methods and interpretation scheme for HLA-DR and DQ typing.

DNA-restriction fragment length polymorphism (RFLP) typing is a well established and standard technique for identification of HLA-DR and DQ allotypes. We present a detailed review of methods for RFLP analysis and a scheme for interpretation of results. This scheme permits the routine identification of HLA-DR and DQ allotypes using the restriction endonucleases TaqI and either HindIII or MspI.     

Similarity of Bovine Rotavirus Receptor and Human Rotavirus Receptor

Rotavirusesaretheleadingcauseofviralgastroenteritis worldwide.Theseviruses,includinghumanrotavirus (HRV)andbovinerotavirus(BRV),whichprimarilyin fectchildrenandyounganimals,cancauseseveredehy dratingdiarrhearesultinginsignificantmorbidity,mortali tyand…     

Deoxyribonucleic acid typing methods for medically important microorganisms.

The ability to type microorganisms to a sub-species level plays an essential role in the diagnosis, treatment and control of human infection. Traditionally, differentiation of microorganisms has involved analysis of phenotypic markers. However, these methods are not universally applicable to all microorganisms, and results may be influenced by environmental factors. Recent developments in DNA analysis, together with the limitations of phenotypic methods, have resulted in an increasing use of procedures based on DNA analysis for the typing of clinically important microorganisms. The aim of this review is to provide an overview of the advantages and disadvantages of the genetic typing techniques currently available.     

Comparison of typing methods for Clostridium difficile isolates.

A simple discriminative typing method for Clostridium difficile has been developed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and restriction enzyme analysis are relatively simple techniques but are difficult to evaluate, especially the restriction enzyme analysis. Immunoblotting and restriction fragment length polymorphism typing facilitate simple discrimination of patterns.     

Comparison of molecular typing methods for Candida albicans.

Four molecular approaches to determining the types of Candida albicans strains were compared. The strains used were those whose repeated DNA (ribosomal and mitochondrial) EcoRI restriction fragment length polymorphisms (RFLP) were determined by Stevens et al. (D. A. Stevens, F. C. Odds, and S. Scherer, Rev. Infect. Dis. 12:258-266, 1990). Scherer and Stevens (S. Scherer and D. A. Stevens, Proc. Natl. Acad. Sci. USA 85:1452-1456, 1988) used the same strains to examine the Southern blots of genomic EcoRI digests probed with the repeated sequence 27A. The results of these investigators were compared with determinations of RFLPs generated from repeated DNA by the enzyme HinfI and examination of the karyotypes of strains under two sets of conditions, one for the smaller chromosomes and one for the larger ones. Analysis of RFLPs of repeated DNA is most convenient but shows the lowest degree of resolution. Use of the repeated sequence and use of karyotype have very high resolution, but the former method is more convenient than the latter. HinfI digestion is more sensitive than EcoRI digestion but equally convenient. By using all four methods, separate types were identified for 18 of the 20 strains examined.     

Evaluation of electrophoretic methods for typing methicillin-resistant Staphylococcus aureus

Three electrophoretic methods of typing methicillin-resistant Staphylococcus aureus (MRSA) strains--plasmid profiles (PP), whole-cell protein profiles (WCPP) and immunoblotting profiles (IP)--were evaluated and compared with phage typing. The results obtained with isolates from 12 outbreaks were compared both within the outbreaks, to determine the consistency of results, and between outbreaks. There was generally good agreement between the typing methods but in only six outbreaks did all four methods indicate the same relationship between isolates. WCPP comprised more than 50 bands; when differences occurred, they were seen in only a few bands. In contrast, IP comprised only one or two major bands and the differences were much easier to interpret. The PPs of many of the isolates were similar; many isolates contained a plasmid of mol. wt (18-25) x 10(6). In several outbreaks both WCPP and IP showed minor differences between isolates that were not apparent with phage typing. When comparisons were made between the 12 index strains and an isolate representing the London epidemic MRSA strain, phage typing and WCPP were the most discriminatory methods; both gave nine distinct patterns, whereas there were eight IPs and only six PPs amongst the 13 strains. It was concluded that both WCPP and IP could provide valuable epidemiological data on MRSA and that IP was the easiest of the three methods to interpret.     

Comparison of six typing methods for Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans is an important pathogen in the etiology of severe periodontitis. For epidemiological studies on the prevalence of certain pathogenic clones and transmission of this bacterium, adequate typing methods are necessary. The purpose of this study was to compare six different typing methods for A. actinomycetemcomitans. Five reference strains and 27 fresh clinical isolates from periodontitis patients were used. Serotyping showed 12 serotype a strains, 13 type b strains, 6 type c strains, and 1 nontypeable strain. Biotyping on the basis of the fermentation of mannose, mannitol, and xylose resulted in six biotypes. Antibiogram typing was evaluated by measuring the inhibition zones of seven antibiotics in agar diffusion tests. With this method eight main types which could be further differentiated into 15 subtypes were found. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of outer membrane proteins were similar among all isolates tested. Restriction endonuclease analysis (REA) of whole chromosomal DNA resulted in five main types. These five main types were further differentiated into 24 subtypes on the basis of DNA fragment differences in the high-molecular-weight region. Hybridization of DNA fragments with ribosomal DNA (ribotyping) resulted in 22 to 24 different types, depending on the restriction endonuclease used. Ribotype patterns were easy to interpret and provided an univocal distinction between different strains compared with REA results. When applied to epidemiologically related isolates, all methods were able to discriminate two clonal types among five isolates from five children from one family. We conclude that serotyping, biotyping, and outer membrane patterns were reproducible but had a low discriminatory potential. REA and ribotyping were reproducible and gave the highest number of distinct types. When the DNA typing methodis were compared, all strains tested could be distinguished. These findings confirm the heterogeneity found within the species A. actinomycetemcomitans.     

Evaluation of three methods for typing herpes simpex viras

Three methods of typing herpes simplex virus were compared. A total of 111 clinical isolates obtained from patients not treated with antivirals and seven resistant mutants selected in vitro were tested by immunofluorescence assay using antibodies against herpes simplex virus types 1 and 2. Twenty-nine isolates were also studied by restriction endonuclease analysis. The sensitivity of isolates and resistant mutants to (E)-5-(2-bromovinyl)-2-deoxyuridine was determined. Although a clear difference between the 50 % inhibitory dose for type 1 and type 2 isolates was observed, some drug-resistant mutants might be misidentified by this method.     

Reproducibility and indices of discriminatory power of microbial typing methods.

When microbial strain-typing methods are compared, the most important characteristics are typeability, reproducibility, and discriminatory power. While typeability and reproducibility can be presented as numerical values, indices of discriminatory power have only recently been described. This paper examines the relationship between reproducibility and indices of discriminatory power. In an individual typing method, an inverse relationship between reproducibility and discriminatory power appears as the number of test differences required in order to distinguish between strains is increased. A method of standardizing the discriminatory power of a typing method to a predetermined reproducibility is presented. In this way the discriminatory powers of different typing methods can be compared while being standardized for the effect of reproducibility.