Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA.

Author(s): Khan G, Kangro HO, Coates PJ, Heath RB

Publication: J Clin Pathol, 1991, Vol. 44, Page 360-5

PubMed ID: 1646235 PubMed Review Paper? No

Purpose of Paper

This paper investigated the inhibitory effects of urine specimens collected from children on the PCR detection of cytomegalovirus (CMV); optimization steps for specimen filtration and PCR analysis were also explored.

Conclusion of Paper

The authors identified urea as the inhibitory component of PCR CMV detection in urine specimens. Urea inhibited PCR analysis at a final concentration of 50 mM or higher. Although urea's inhibitory activity was circumvented by specimen dialysis or ultrafiltration prior to PCR analysis, amplicon intensity was reduced necessitating a two stage nested PCR approach.

Studies

Study Purpose

The purpose of this study was to identify the urine component that inhibited PCR detection of CMV.

Summary of Findings:

The authors report that urea is a complete inhibitor of PCR at a concentration of 100 mM and partially inhibitory at a concentration of 50 mM. The inhibitory activity of urine was independent of creatinine and electrolyte concentrations. Two neonatal specimens with extremely low urea concentrations (10 mM) that were reactive for glucose also resulted in inhibition of the PCR reaction.

Biospecimens

Bodily Fluid - Urine

Preservative Types

Frozen

Diagnoses:

Not specified

Platform:

Analyte	Technology Platform
DNA	PCR

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Biospecimen Aliquots and Components	Biospecimen components	Urea concentration (5-250 mM) Creatinine concentration Na+ concentration K+ concentration Glucose concentration

Study Purpose

The purpose of this study was to identify purification and analytical methods that circumvent the inhibitory effect of urine for PCR analysis.

Summary of Findings:

While the authors report that both dialysis and ultrafiltration of urine specimens were successful in the removal of inhibitory activity, Millipore cups were preferred due to benefits associated with volume, time, and financial requirements. Specimen filtration, however, reduced amplicon intensity, which was circumvented through the use of a nested two stage PCR approach that increased sensitivity 10-fold compared to conventional one stage PCR.

Biospecimens

Bodily Fluid - Urine

Preservative Types

Frozen

Diagnoses:

Not specified

Platform:

Analyte	Technology Platform
DNA	PCR

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Analyte Extraction and Purification	Analyte purification	Dialysis Centricon-100 ultrafiltration tubes Millipore ultrafiltration cups
PCR Specific	Technology platform	One stage PCR Two stage nested PCR

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