The use of bovine serum albumin to improve the RT-qPCR detection of foodborne viruses rinsed from vegetable surfaces

Abstract

Aims: To demonstrate that produce rinsates used for RT‐qPCR detection of foodborne viruses may cause significant PCR inhibition and propose a means to reduce its impact on sensitivity. Methods and Results: Here, it is shown that rinsing and concentration from spinach and precut lettuce have the potential to generate RNA extracts that are inhibitory to RT‐qPCRs assembled from commercial kits for the detection of norovirus GII (NoV GII), hepatitis A virus (HAV), hepatitis E virus (HEV), rotavirus (RV) and feline calicivirus (FCV) as sample process control. It is further shown that the addition of bovine serum albumin (BSA) to those reactions restored a positive signal in all cases. The effect of BSA was dependent upon the primer/probe combination. Moreover, two of the detection systems (FCV and HAV) strongly benefited from the addition of BSA even in the absence of PCR inhibitors. Conclusions: BSA was shown to restore positive signals in five different RT‐qPCR systems that were otherwise completely inhibited by produce rinsate extracts. It is therefore suggested to consider the addition of BSA to RT‐qPCRs for the detection of foodborne viruses when inhibition is observed. Significance and Impact of the Study: This study clearly demonstrates the potency of PCR inhibitors generated during routine virus concentration from produce and that it can be alleviated by the addition of BSA to the RT‐qPCRs. Although used elsewhere, the addition of BSA to PCRs is not a common practice in this growing field of research.

Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR

“…Additional procedures have been developed and are still being improved, for the removal or reduction of the effects of PCR inhibitors such as tannic, humic and fulvic acids which are abundant in natural waters (Kreader 1996;Al Soud and Radstrom 2000;Guy et al 2003;Skotarczak 2009;Plante et al 2011;Schriewer et al 2011). In our studies, the differences between the nested PCR signals and C T values obtained for the DNA of oocysts recovered from lake and from distilled water implicate the presence of inhibitors in environmental water.…”

“…Many chemicals are used to deactivate PCR inhibitors, e.g., acetamide, betaine, dextran, glycerol, formamide, pectinase, polyvinylpyrrolidene, aqueous solutions of non-fat dry milk or bovine serum albumin (BSA) (Al Soud and Radstrom 2000;Plante et al 2011). Earlier studies revealed that BSA is one of the most efficient facilitators of conventional PCR (Al Soud and Radstrom 2000;Skotarczak 2009;Plante et al 2011;Schriewer et al 2011).…”

“…Earlier studies revealed that BSA is one of the most efficient facilitators of conventional PCR (Al Soud and Radstrom 2000;Skotarczak 2009;Plante et al 2011;Schriewer et al 2011). Bovine serum albumin binds to many substances and prevents their binding to Taq DNA polymerase, target DNA and magnesium ions (Kreader 1996;Plante et al 2011).…”

“…In our studies, the differences between the nested PCR signals and C T values obtained for the DNA of oocysts recovered from lake and from distilled water implicate the presence of inhibitors in environmental water. The reduction or removal of PCR inhibitors is an essential component in the molecular detection of microorganisms in environmental samples, because inhibitors may interfere with the cell lysis step, inactivate the thermostable DNA polymerase and/or interfere with nucleic acids (Al Soud and Radstrom 2000;Plante et al 2011;Schriewer et al 2011). The reduction of PCR inhibitors before, during, or after DNA extraction has become an important step in the molecular diagnosis of protozoan pathogens in water and other environmental samples (Jiang et al 2005).…”

“…Many studies have shown that PCR inhibitors occurring in water samples suppress or reduce PCR amplification (Johnson et al 1995;Loge et al 2002). Environmental samples (water and soil), as well as stool samples are rich in PCR inhibitors which can be co-extracted with DNA during the DNA isolation and purification process and which can interfere with the PCR amplification (Kreader 1996;Al Soud and Radstrom 2000;Jiang et al 2005;Plante et al 2011;Schriewer et al 2011).…”