Establishment Real-time PCR for Detection Noroviruses in Stool Specimens

Noroviruses are usually detected in stool specimens. Such samples commonly contain components reported to be (RT-) PCR inhibitors as well as the main inhibitory substance is the reverse transcriptase enzyme itself. This study provides a complex overview of the influence of RT systems, on the efficiency of PCR to detect Norovirus in stool specimens. The efficiency of three commonly used reverse transcriptases, namely Moloney Murine Leukemia Virus (M-MuLV), Avian Myeloblastoma Leukemia Virus (AMV) and Improm II reverse transcriptase (RT), have been compared to RT-PCR. The study used total RNA extracted from the same stool sample, no bias could therefore be attributed to sample preparation or host individuality. All amplification reactions were run using starting material from the stock, 1/10, 1/100 and 1/1000 cDNA dilutes. In the results, Improm II yielded very weak bands, in the presence of the stock cDNA and completely failed to amplify DNA in the presence of the 1/10, `/100, 1/1000 cDNA dilute. Whereas AMV yielded bands in the presence of the stock and 1/10 cDNA dilute and M-MuLV yielded bands in the presence of the stock and all cDNA dilutes.


Background
Noroviruses are a major cause of epidemic and sporadic gastroenteritis in humans and clinical signs and lesions of gastroenteritis were reported in bovines. The full-length sequencing of different human Norovirus genomes has allowed the development of reverse transcription polymerase chain reaction (RT-PCR) which has become the gold standard for Norovirus diagnosis in stool and environmental samples (Atmar and Estes, 2001;Jiang et al., 1993;Lambden et al., 1993). With such samples, the control for inhibition of the reaction during amplification and detection is crucial to avoid false negative results, which might otherwise not be detected (Scipioni et al., 2008).
For RNA detection, the most fundamental step is the in-vitro reverse transcription (RT) of RNA to its complementary DNA (cDNA), catalyzed by the enzyme reverse transcriptase (Arezi and Salehi, 2006;Kawasaki, 1990;Sellner et al., 1992;Verma, 1997). Many factors such as the quality of the RNA, the presence of inhibitor co-extracted with the RNA, as well as the RT efficiency are known to affect the total yield of cDNA (Bustin and Nolan, 2004;Arezi et al., 2010). Some studies have shown the main inhibitory substance is the reverse transcriptase enzyme itself (Chandler et al., 1998;Chumakov, 1994). As such, the enzyme needs to be removed or denatured directly after the RT reaction is complete to reduce its inhibitory and/or nuclease activities. This can be achieved via a heat or an alternative inactivation step, which has been incorporated in almost all commercially available reverse transcriptase kits/systems. However, with the increasing number of heat-resistant RT enzymes (Arezi et al., 2010), and the fact that many RT enzymes retain their inhibitory properties even post denaturation (Suslov and Steindler, 2005), there is a need for alternative strategies to overcome this limitation. Dilutions of RT products (cDNA) are often used to minimize post-RT inhibitory effects (Sellner et al., 1992;Levesque et al., 2007;Chandler et al., 1998;Chumakov, 1994;Wilson, 1997), although this has the negative consequence of reducing quantification precision, which is especially problematic when dealing with very low copy templates (Okello et al., 2010).
Evaluating the capacity and detection limits of different commercial RT systems in regard to these conditions is fundamental to maximizing the sensitivity of the RT-PCR .
For that reason, for detection Norovirus in stool samples the first stage of gene expression analysis, the RT step, was examined in order to give further directives about the appropriate choice of the RT system for RT-PCR assay.
The aim of this work was to study the sensitivity of some commercially available reverse transcriptases which able to detect Noroviruses circulating in the human and bovine populations.

Agarose gel electrophoresis
A PCR reaction was considered positive if a 319 bp band could be visualized on an agarose gel following electrophoresis; however, in many instances DNA amplification success (band intensity) was a continuum, decreasing as the inhibitor concentration increased.

AMV reverse transcriptases (RT)
For the RT-PCR amplification of cDNA, the AMV reverse transcriptases were able to produce bands from stock and 1/10 diluents ( Figure 1).

Improm II reverse transcriptases (RT)
Improm II reverse transcriptases could only produce a trace amount of stock cDNA amplification products ( Figure 2).

M-MuLV-Reverse Transcriptase (RT)
For the RT-PCR amplification of cDNA, M-MuLV reverse transcriptases were able to produce bands from stock and all cDNA dilutions ( Figure 3). We demonstrated that the use of M-MuLV reverse transcriptases was superior to AMV and Improm II reverse transcriptase for the synthesis of Noro virus cDNA from stool samples. We show that reverse transcription yields vary up to 1000 fold with the M-MuLVreverse Transcriptase.

Discussion
Feces constitute complex biological samples, which cause problems when PCR is used as a diagnostic method, not only because of the presence of numerous types of bacteria but also because of the different kinds of food, degradation products present (Greenfield and White, 1993).
Messenger RNA profiling relies heavily on a reverse transcription step, which is accompanied by PCR. The recombinant Moloney murine leukemia virus M-MuLV, Improm II and avian myeloblastosis virus AMV-RT, are the most frequently used reverse transcriptases for this purpose. There is a well-known inhibitory effect of PCR by RT components (Sellner et al., 1992;Fehlmann et al., 1993;Chumakov, 1994;Chandler et al., 1998;Liss, 2002). The introduction of micrograms of RNA into the RT step, followed by the extensive dilution of RT reaction before PCR execution, is supposed to minimize this inhibition (Sellner et al., 1992;Chumakov, 1994;Chandler et al., 1998;Aatsinki et al., 1994;Karsai et al., 2002). These conditions often are not met for two reasons: there is not enough RNA available, and the extensive dilution will negatively affect the precise detection of rarely expressed genes (Suslov and Dennis, 2005).
In this study, we addressed the efficiency of different commercial RTases and assessed their sensitivity in their respective systems, following the vendors' recommendations. We found that, whereas the RNA-to-cDNA conversion of abundant transcripts is efficient with all commercial RT systems tested in the presence of various amounts of background RNA, cDNA could not be detected with Improm II-RT systems when high cDNA diluents are involved. We found that the RT step is critical for gene analysis and that a judicious choice is required when starting RNA material is limited.
In this study, M-MuLV reverse transcriptases were able to produce bands from stock and all diluents cDNA. Loisy et al (2005)  In this study, AMV was able to produce bands from stock and 1/10 cDNA. The addition of BSA, that is able to scavenge a variety of inhibitory substances (Kreader, 1996), (Wise et al., 2003) and Trujillo et al (2006) used Avian myeloblastosis virus (AMV) super reverse transcriptase (Molecular Genetic Resources, Tampa, FL) for Norovirus detection after RNA extraction from stool samples. Jiang et al (1999) used AMV-RT enzyme after RNA extraction from stool samples. Richards et al (2004) used DuraScript reverse transcriptase (enhanced avian myeloblastosis virus) for Genogroup I and II Noroviruses Detected in Stool Samples.
Christian Beuret (2003) used Sensiscript-RT kit for detection of Norovirus genogroup in stool samples. Hoehne and Schreier (2006) used Omniscript and Sensiscript reverse transcriptases for detection of Norovirus genogroup I and II in stool samples.
In conclusion, these enzymes differed significantly in their efficiency of cDNA synthesis as indicated by the degree of amplification by PCR; Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase produced greater amplification using RT-PCR than that of the Avian Myeloblastoma Leukemia Virus (AMV) and Improm II reverse transcriptase.

Stool samples and RNA extraction
The PCR assay was optimized using archived positive human stool sample, a stool suspension 10% in sterile phosphate-buffered saline (PBS) was performed, RNA was extracted from PBS suspension with the QIAamp viral RNA mini kit (QIAGEN, Hilden, Germany) according to the column centrifugation procedure described by the manufacturer. The amount of RNA in the extracted material was measured using a NanoDrop spectrophotometer.

DNase treated RNA
To reduce nonspecific amplification and to prevent carryover contamination by Norovirus cDNA, RNA was treated with DNase I before RT, for this viral RNA (12.5 µL) was added to the reaction mixture (2.5 µL) containing DNase Ⅰ buffer and 1 U of RQ1 DNase (Promega). The reaction at 7 mixture was incubated at 37℃ for 30 min to digest DNA and then 5℃ for 5 min to inactivate the enzyme.

Reverse TRANSCRIPTION (RT)
To address the efficiency of commercial RT systems, we set up an RT-PCR system that could independently monitor the influence of reverse transcriptase. The commercial RT systems tested were Improm II, AMV and M-MuLV-Reverse Transcriptase (RT). The study used total RNA extracted from the same sample and method. No bias could therefore be attributed to sample preparation or individuality.

Improm II reverse transcription (RT)
The reverse transcription was performed in two steps. For the first step, 3 µL of RNA template was mixed with 6 µL nuclease-free water and 1 µL Random Hexamers primer and incubated at 70℃ for 5 min followed by cooling to 4℃using a thermal cycler. For reverse transcriptase (RT) step, a total volume of 20 µL reaction mixture was prepared consisting of 10 µL RNA/primer mixture from the first step, 4 µL Improm II 5× Reaction Buffer, 2.4 µL 25 mM MgCl 2 , 1 µL dNTPs, 0.6 µL nuclease-free water, 1 µL RNease inhibitor, 1 µL Improm II reverse transcriptase. The mixture was returned to the thermal cycler and incubated at 25℃ for 5 min, 42℃ for 60 min and 70℃ for 15 min before being cooled to 4℃.

cDNA suspension
All amplification reactions were set up using a series concentration of cDNA in nuclease-free water. Stock, 1/10, 1/100 and1/1000 suspensions were carried out.