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PCR inhibition in stool samples in relation to age of infants
Sami Oikarinen1, Sisko Tauriainen, Hanna Viskari
1JDRF Center for the Prevention of Type 1 Diabetes in Finland, Finland. Sami.Oikarinen@uta.fi
Insights
PCR inhibitors are common in children's stool samples, potentially causing false negatives. Adding bovine serum albumin (BSA) effectively neutralizes these inhibitors, ensuring accurate PCR testing.
Area of Science:
- Molecular Biology
- Clinical Diagnostics
- Pediatric Gastroenterology
Background:
- Polymerase Chain Reaction (PCR) is increasingly used in diagnostic virology.
- PCR inhibitors in clinical samples can lead to inaccurate false-negative results.
Purpose of the Study:
- To investigate the prevalence of PCR inhibitors in stool samples from children aged 3 to 24 months.
- To explore the relationship between age, diet, and inhibitor presence.
- To assess methods for overcoming PCR inhibition.
Main Methods:
- Stool samples were collected from children aged 3-24 months.
- Extracted RNA was spiked with Semliki Forest Virus RNA and amplified via PCR.
- Inhibition was quantified by comparing amplification rates to controls.
- Dietary influence was assessed by comparing samples from exclusively breastfed vs. non-exclusively breastfed infants.
- The efficacy of bovine serum albumin (BSA) in mitigating inhibition was tested.
Main Results:
- 12% of samples showed complete and 19% partial PCR inhibition.
- Inhibition was not detected in infants under 6 months but present in 17% of older infants (6-24 months).
- Breastfeeding was more prevalent in younger infants.
- Addition of BSA completely eliminated inhibitory effects, yielding positive results for all samples.
Conclusions:
- PCR inhibitors are frequently found in children's stool samples.
- Dietary components may contribute to PCR inhibition.
- BSA is a simple and effective agent for neutralizing stool-derived PCR inhibitors, improving diagnostic accuracy.
Background:
PCR is rapidly replacing traditional methods in diagnostic virus laboratories. PCR inhibitors, which are often present in clinical samples, may lead to false negative test results.
Objectives:
The aim was to study the presence of PCR inhibitors in stool samples collected from 3- to 24-month old children.
Study Design:
Total RNA fraction extracted from stool samples was spiked with a standardized amount of Semliki Forest Virus RNA and amplified using specific PCR primers. The presence of PCR inhibitors was detected by a decrease in amplification rate compared to spiked water samples. Inhibition in different age groups and dietary origin of PCR inhibitors were analyzed by comparing the samples taken during exclusive and non-exclusive breastfeeding periods. The inactivation of PCR inhibitors was also assessed.
Results:
Complete inhibition was seen in 12% (13/108) and partial inhibition in 19% (21/108) of the samples. Inhibition was seen in none of the stool samples (0/31) taken from infants younger than 6 months compared to 17% of samples (13/77) taken from 6 to 24 months old infants (p<0.036). Breastfeeding was more common in younger age group. Addition of bovine serum albumin (BSA) into the reaction mixtures eliminated the effect of inhibitors leading to all samples being positive.
Conclusions:
PCR inhibitors are frequent in stool samples. They may originate from dietary components and can lead to false negative PCR results. The addition of BSA to the cDNA and PCR reactions proved to be an easy and effective method for eliminating the inhibitory effect of these compounds.
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