Detection of virulence genes in Malaysian Shigella species by multiplex PCR assay.
BACKGROUND
In Malaysia, Shigella spp. was reported to be the third commonest bacterial agent responsible for childhood diarrhoea. Currently, isolation of the bacterium and confirmation of the disease by microbiological and biochemical methods remain as the "gold standard". This study aimed to detect the prevalence of four Shigella virulence genes present concurrently, in randomly selected Malaysian strains via a rapid multiplex PCR (mPCR) assay.


METHODS
A mPCR assay was designed for the simultaneous detection of chromosomal- and plasmid-encoded virulence genes (set1A, set1B, ial and ipaH) in Shigella spp. One hundred and ten Malaysian strains (1997-2000) isolated from patients from various government hospitals were used. Reproducibility and sensitivity of the assay were also evaluated. Applicability of the mPCR in clinical settings was tested with spiked faeces following preincubation in brain heart infusion (BHI) broth.


RESULTS
The ipaH sequence was present in all the strains, while each of the set1A, set1B and ial gene was present in 40% of the strains tested. Reproducibility of the mPCR assay was 100% and none of the non-Shigella pathogens tested in this study were amplified. The mPCR could detect 100 colony-forming units (cfu) of shigellae per reaction mixture in spiked faeces following preincubation.


CONCLUSIONS
The mPCR system is reproducible, sensitive and is able to identify pathogenic strains of shigellae irrespective of the locality of the virulence genes. It can be easily performed with a high throughput to give a presumptive identification of the causal pathogen.
Thong
Kwai Lin
KL
Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. thongkl@um.edu.my
Hoe
Susan Ling Ling
SL
Puthucheary
S D
SD
Yasin
Rohani  Md
R
D000942:Antigens, Bacterial / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers / Q000737:chemistry; D004405:Dysentery, Bacillary / Q000175:diagnosis / Q000382:microbiology; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005243:Feces / Q000382:microbiology; D005787:Gene Frequency / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012760:Shigella / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity*; D037521:Virulence Factors / Q000235:genetics* / Q000302:isolation & purification*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D017931:DNA Primers; D037521:Virulence Factors; C063500:ipaH protein, Shigella flexneri