PCR detection of Giardia lamblia in stool: targeting intergenic spacer region of multicopy rRNA gene.
A PCR based detection that amplifies the 552-bp intergenic spacer (IGS) region of multicopy rRNA gene of Giardia lamblia and 320-bp internal sequences to first PCR product has been used in diagnosis of giardiasis in stool sample. The primers were found highly specific to Giardia spp. only, because no amplification was observed with DNAs from other enteric pathogens like Escherichia coli, Shigella dysenteriae and Entamoeba histolytica. The test could detect even less than 2 pg of genomic DNA from Giardia trophozoites. In direct diagnosis of Giardia lamblia in stool samples, it was observed that PCR amplification of IGS followed by nested PCR could enhance the sensitivity and specificity of the tests manifold and the system was able to detect as low as 10 parasites in 100 microl of stool. The comparative evaluation of the present system with conventional microscopy, CIEP and ELISA in the diagnosis of giardiasis from diarrhoeic stool samples and control subjects demonstrated a 100% correlation among nested PCR, microscopic examination and ELISA in patients suggestive of giardiasis (Group I) and control subjects (Group II). In Group I cases (patients suffering from other than giardiasis), CIEP, ELISA and nested PCR showed better results than microscopic examination. However, among them, PCR was found most sensitive and specific because 20% positivity was noticed by PCR whereas CIEP and ELISA showed only 7.14% and 12.85%, respectively. Break-up results showed that all the samples which were positive by CIEP or ELISA, also found positive by PCR. The present observation clearly suggests the use of PCR that amplifies the intergenic spacer region of multicopy rRNA gene of Giardia lamblia followed by nested PCR for routine, quick and reliable detection of Giardia lamblia in stool samples.
Ghosh
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S
Department of Biochemistry and Dr B. C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, India.
Debnath
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A
Sil
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De
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Chattopadhyay
D J
DJ
Das
P
P
D000818:Animals; D003378:Counterimmunoelectrophoresis; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000032:analysis* / Q000235:genetics; D004275:DNA, Ribosomal / Q000032:analysis / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005243:Feces / Q000469:parasitology*; D020459:Genes, rRNA / Q000235:genetics*; D016829:Giardia lamblia / Q000235:genetics* / Q000302:isolation & purification*; D005873:Giardiasis / Q000175:diagnosis* / Q000469:parasitology; D006801:Humans; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013997:Time Factors
D017931:DNA Primers; D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D015345:Oligonucleotide Probes