DIAGNOSTYKA MOLEKULARNA
W ZAKAŻENIACH SZPITALNYCH
Beata Krawczyk
Katedra Mikrobiologii Wydziału Chemicznego Politechniki Gdańskiej
ul. G. Narutowicza 11/12, 80-952 Gdańsk
Wpłynęło w kwietniu 2007 r.
1. Wstęp.
2. Metody genotypowe różnicowania drobnoustrojów.
2.1. Analiza
restrykcyjna chromosomalnego DNA połączona z elektroforezą pulsową
(REA-PFGE). 2.2. Analiza profili plazmidowych.
2.3. Hybrydyzacja
DNA-DNA. 2.4. Metody oparte o amplifikację DNA techniką PCR.
2.4.1. Amplifikacja znanych regionów genomu połączona z
analizą restrykcyjną
(PCR-RFLP). 2.4.2. Wykorzystanie sekwencji repetytywnych w
genotypowaniu bakterii. 2.4.3. PCR fingerprinting.
2.4.4. Sekwencjonowanie genów. 2.5. Techniki oparte
o ligację adaptorów
oligonukleotydowych. 2.5.1. AFLP (Amplified Fragment Length
Polymorphism). 2.5.2. IRS-PCR (Infrequent Restriction Site
PCR). 2.5.3. ADSRRS (Amplification of DNA Surrounding Rare
Restriction Sites). 2.6. Metody oparte na właściwościach
topnienia DNA. 2.6.1. Elektroforeza w gradiencie
denaturującym (DGGE - Denaturing Gradient Gel
Electrophoresis; TGGE - Temperature Gradient Gel Electrophoresis).
2.6.2. Technika SSCP (Single Strand Conformation
Polymorphism). 2.6.3. PCR MP (PCR Melting Profiles).
2.6.4. Real-time PCR. 3. Wnioski końcowe
Molecular diagnostic in hospital infections
Abstract:
In this review, we have compared some of the most current technologies
for genotyping with regard to the principles of the methods and their
reproducibility, discrimination power, ease of use and cost.
Macrorestriction analysis of genomic DNA followed by pulsed-field gel
electrophoresis (REA-PFGE) has become the "gold standard" for molecular
typing. However, REA-PFGE is time-consuming and labor-intensive and can
be performed only in reference laboratories with skillful
technicians.
Due to these drawbacks, this typing method is not ideal for
health
departments undertaking routine analysis of large numbers of isolates.
A variety of PCR-based methods for displaying DNA sequence
polymorphism
have been developed. Some of the methods such as RAPD, AFLP and ADSRRS
regarded as alternative to REA-PFGE, are useful systems for molecular
typing of microorganisms.
1. Introduction.
2. Genotyping methods for the differentiation of
microorganisms. 2.1. Macrorestriction analysis of
DNA-fragments using
pulsed-field gel electrophoresis (REA-PFGE). 2.2. Plasmid
profile
analysis. 2.3. DNA-DNA hybridization. 2.4. Methods
based on PCR
amplification. 2.4.1. Amplification of known DNA regions with
restriction analysis (PCR-RFLP). 2.4.2. Usefulness of repetitive
sequences in genotyping. 2.4.3. PCR fingerprinting.
2.4.4. Sequencing.
2.5. Techniques based on oligonucleotide adapters ligation.
2.5.1. AFLP
(Amplified Fragment Length Polymorphism). 2.5.2. IRS-PCR
(Infrequent
Restriction Site PCR). 2.5.3. ADSRRS (Amplification of DNA
Fragments
Surrounding Rare Restriction Sites). 2.6. Methods based on
melting
properties of DNA. 2.6.1. Electrophoresis in denaturing
gradient (DGGE
- Denaturing Gradient Gel Electrophoresis; TGGE - Temperature Gradient
Gel Electrophoresis). 2.6.2. SSCP technique (Single Strand
Conformation
Polymorphism). 2.6.3. PCR MP (PCR Melting Profiles).
2.6.4. Real-time
PCR. 3. Summary
Słowa kluczowe: epidemiologia,
diagnostyka molekularna, epidemia,
genotypowanie, PCR
Key words: epidemiology, molecular
diagnostic, outbreak, genotyping, PCR
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