A SYBR Green RT-PCR assay in single tube to detect human and bovine noroviruses and control for inhibition
© Scipioni et al; licensee BioMed Central Ltd. 2008
Received: 06 May 2008
Accepted: 14 August 2008
Published: 14 August 2008
Noroviruses are single-stranded RNA viruses belonging to the family Caliciviridae. They are a major cause of epidemic and sporadic gastroenteritis in humans and clinical signs and lesions of gastroenteritis were reported in bovines. Due to their genetic proximity, potential zoonotic transmission or animal reservoir can be hypothesized for noroviruses. RT-PCR has become the "gold standard" for the detection of noroviruses in faecal and environmental samples. 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. The aim of the reported method is to detect, with a SYBR Green technology, a broad range of noroviruses with a control for inhibition.
A SYBR Green real-time RT-PCR assay was developed making use of a foreign internal RNA control added in the same tube. This assay is able to detect human and bovine noroviruses belonging to genogroups I, II and III and to distinguish between norovirus and internal control amplicons using melting curve analysis. A 10-fold dilution of samples appears to be the method of choice to remove inhibition. This assay was validated with human and bovine stool samples previously tested for norovirus by conventional RT-PCR.
This SYBR Green real-time RT-PCR assay allows the detection of the most important human and bovine noroviruses in the same assay, and avoids false negative results making use of an internal control. Melting curves allow the discrimination between the internal control and norovirus amplicons. It gives preliminary information about the species of origin. The sensitivity of the developed assay is higher than conventional RT-PCR and a 10-fold dilution of samples showed a better efficiency and reproducibility to remove RT-PCR inhibition than addition of bovine serum albumin.
Norovirus is one of the four genera currently accepted into the family Caliciviridae. Other genera in this family include Sapovirus, which causes gastroenteritis in humans, as well as Lagovirus and Vesivirus, neither of which are pathogenic for humans. Noroviruses are small, non-enveloped viruses with a diameter of approximately 27–35 nm. They have a positive-sense, single stranded RNA genome . Norwalk virus, the prototype strain of the genus norovirus, was first described in 1972 in association with an outbreak of gastroenteritis and vomiting involving children and staff at an elementary school in Norwalk, Ohio .
Noroviruses are now recognized as a common cause of human infectious gastroenteritis in all age groups, especially in restaurants and institutions such as nursing homes and hospitals [3–5]. They are one of the main causes of foodborne gastroenteritis [6, 7]. Furthermore, several animal noroviruses genetically closely related to human noroviruses have been recently discovered [8–10]. Their existence raises important questions about animal reservoirs and potential zoonotic transmission . The diagnostic of human and bovine noroviruses is impaired by the difficulties to replicate it in cell culture , although a tridimensional culture system was recently shown to be able to grow human noroviruses . The full-length sequencing of different human norovirus genomes has allowed the development of reverse transcription polymerase chain reaction (RT-PCR) [13, 14], which has become the gold standard for norovirus diagnosis . Due to the genetic diversity among noroviruses, it is very difficult to find an appropriate primer pair that is both sensitive and specific for detection of all noroviruses. The most conserved region of the genome is the RNA polymerase gene and several primer pairs have been selected in that region , as the one used in this assay . Real-time RT-PCR assays are more and more developed and has become the method of choice for the detection and the characterization of norovirus. Many different real-time RT-PCR assays for norovirus genogroups I and II had been developed [17–19] and co-detection of human and animal noroviruses was described in a multiplex assay  or simultaneously .
Noroviruses are usually detected in clinical specimens (faeces and vomit) and contaminated food, water or sewage [22–25]. Such samples commonly contain components reported to be (RT-)PCR inhibitors [26, 27], leading to a high risk of false negative results or a decrease of the Ct value. A control to adequately detect problems with either RNase contamination or RT-PCR inhibitors is necessary to avoid false-negative responses for samples submitted for diagnosis [28, 29]. An internal control is crucial to diagnostic (RT-)PCR assays. It is co-amplified with the target sequence and a negative result indicates a total (RT-)PCR failure. Also partial decrease of amplification capability can be estimated compared with the decrease of the internal control Ct value (internal control in the sample versus internal control alone).
The aim of this study was the development of a SYBR Green real time RT-PCR method able to detect the most important genogroups of noroviruses circulating in the human and bovine populations. This assay includes an internal RNA control and has been designed and validated for the diagnosis of noroviruses in human and bovine stool samples. Melting curve analysis allows the distinction between the internal control and norovirus amplicons and gives some indication about the species of origin. Moreover, the use of this single tube assay, cheaper than a TaqMan analysis, has the great advantages to detect (RT-)PCR inhibition that may lead to false negative results.
Validation of the SYBR Green real-time RT-PCR assay – control of inhibition
The set up of the internal RNA control had been previously described  and the primers used in the SYBR Green real-time RT-PCR had been validated for their specificity by Vennema and collaborators .
Serial dilution of the norovirus internal RNA control transcribed in vitro demonstrated that 2 μl of the 109 fold dilution contained adequate template to produce a detectable product by melting curve analysis following real-time RT-PCR. This corresponds to a quantity of 1.9 × 10-6 ng of internal RNA control or 5,800 copies .
Analysis of human and bovine stool samples
Comparison of the detection of human and bovine noroviruses by the conventional RT-PCR assay and the SYBR Green assay
SYBR Green RT-PCR
The use of the internal control for norovirus real-time RT-PCR diagnosis in 86 stool samples identified inhibition of RT-PCR in 32.6% of stool samples tested in this study (Table 1). Two different methods were used on extracted RNA from samples showing inhibition. One is a 10-fold dilution of the extracted RNA before testing with the SYBR Green assay and the second one is the addition of bovine serum albumin (BSA) in the mix. Among the 28 samples showing inhibition, a 10-fold dilution was active in all samples compared to BSA that failed to remove inhibition from 4 samples.
Considering the SYBR Green assay as the gold standard (Table 1), the relative sensitivity of the conventional RT-PCR used in this study was 92.6% (95% confidence interval (CI) 82.1–97.9). The relative specificity was 100% (CI 91–100) for all techniques. Confirming these results, Kappa value showed a high level of agreement between conventional RT-PCR and the SYBR Green real-time RT-PCR (Kappa value: 0.90; CI 0.81–0.99).
In this study we developed a sensitive and broadly reactive real-time SYBR Green RT-PCR assay that interestingly detects human genogroups I and II and bovine genogroup III noroviruses in a single tube, including an internal control. This assay takes into account that samples may contain inhibitors of PCR or RT-PCR [22, 23]. The method uses one-step, hot start RT-PCR with thermostable DNA polymerase. The one step protocol simplifies the method and reduces the risk of contamination of RNA. Moreover it is useful for routine diagnosis as there is no post-amplification processing of the product.
The genetic diversity of noroviruses makes though to select a pair of primers capable of detecting all the different norovirus genogroups. The commonly used JV12-13 primer pair has been replaced by JV12Y-13I, which contains degenerated bases to allow the detection of a larger panel of noroviruses . This primer pair is able to detect noroviruses belonging to human genogroups I and II and bovine noroviruses belonging to genogroup III . The variability in the melting temperature between human and bovine noroviruses can be also explained by the genetic diversity among norovirus sequences [31, 32], even if the amplicon is located in the polymerase region highly conserved among noroviruses. Therefore, this assay has the rare advantage to detect norovirus strains irrespectively of their origin (human or bovine). There are few methods described that allow the detection of both human and bovine noroviruses. Compared to the method recently published by Wolf and collaborators , our assay has the advantage to detect noroviruses in the same reaction tube and to control for inhibition of the reaction. It is also to detect mixed infection (presence of human and bovine noroviruses in the same sample), by the presence of two distinct peaks.
A common problem with RT-PCR is the presence of (RT)-PCR inhibitors which may cause false negative results. Therefore, to avoid such false negative results, the internal RNA control set up previously  was used in this real-time SYBR Green RT-PCR assay. It was synthesized in vitro from a foreign DNA template, in order to decrease interference with norovirus amplicons [33, 34]. It has the advantages of representing no risk for human or animal health because it does not contain any infectious material, and being stable compared to live control viruses that can evolve and change during their replication. The norovirus amplification is favored compared to the internal control because the RT-PCR of the later results in a larger product. It is an essential property for its function. It means a decrease of the Ct value of the internal control if noroviruses are present in the sample.
With this internal RNA control, inhibition can be detected without the need of additional primer pairs or an additional reaction run for this purpose and the effective identification of samples containing endogenous inhibitors of RT-PCR is allowed. This improvement is crucial for early intervention and control in norovirus outbreaks.
With routine samples used for diagnosis, 36.8% of human samples and 24.1% of bovine samples showed presence of inhibition that can vary a lot among samples and may depend on the type of sample but also on the intrinsic characteristics of the sample (for example, herbivorous or omnivorous diet). When inhibition was detected in samples, the extracted RNA was tested a second time with the SYBR Green assay, on 10-fold diluted RNA or with BSA added in the RT-PCR mix. Sample dilution is often effective as the inhibitory factors can be diluted out, however, enough quantities of target nucleic acid must be present in order to be detected after dilution . The addition of BSA, that is able to scavenge a variety of inhibitory substances , does not have this inconvenience. Our experience in using those two techniques to remove inhibitors led to the conclusion that a 10-fold dilution is more efficient and reproducible than the addition of BSA.
In conclusion, the real-time assay described in this study is an accurate, sensitive, specific and quick method for the detection of a wide range of noroviruses belonging to genogroups I, II and III. At the same time it offers a method to detect samples containing inhibitors, avoiding false negative results by using an internal control. This assay will be applicable to clinical diagnosis in human and animal laboratories, detection of viruses in food or environmental samples. It is the first SYBR Green real-time assay that uses a single primer pair able to detect human and bovine noroviruses simultaneously. A 10-fold dilution of RNA appears to be the method of choice to remove inhibition.
The melting curve analysis gives presumption for the virus origin regarding the host and points out interesting samples to sequence for further studies (bovine norovirus with a melting temperature similar to the human norovirus ones). This property is of upmost importance regarding classification and study of transmission routes of noroviruses. This requires sequencing step in addition to detection. Although neither a zoonotic transmission, nor identification of an animal reservoir of norovirus have been already identified in natural condition, experimental evidence of cross infection was provided with successful inoculation of pigs with human norovirus [8, 37]. Moreover bovine noroviruses have been detected in the food chain, in a bivalve mollusc sample which was contaminated with human noroviruses . This increases the risk of crossing the species barrier and the probability of the emergence of recombinant viruses. In that context, a diagnostic assay that has the capacity to detect both human and bovine noroviruses is of high interest.
Human and animal stool specimens
Fifty seven human and 29 bovine stool samples were tested. Human samples were selected from faeces collected over a 2-year period (2000–2002) by the Medical Microbiological and Virological Laboratory of the University hospital of Liege and from outbreaks in Belgium provided in part by the Institute for Public Health in Brussels and the Virology Laboratory of the St Luc University hospital (2006–2007). Bovine samples were taken from faeces collected by the regional animal diagnostic laboratories "ARSIA" (Association Régionale de Santé et d'Identification Animales) in Belgium over a two year period (2002–2003). All bovine and human samples had been tested previously for norovirus by conventional one-step RT-PCR and sequenced for confirmation. The stool specimens were stored at -80°C. All positive samples were used in this study and 36 negative samples were randomly selected.
Processing of stool samples, RNA extraction and conventional RT-PCR
The all procedure was already described . Briefly, stool samples were 10-fold diluted in phosphate-buffered saline and RNA was extracted using the QIAamp viral RNA Mini Kit (Qiagen, Leusden, The Netherlands). A one-step RT-PCR kit, the Access RT-PCR System (Promega, Leiden, The Netherlands), was used with broadly reactive primer pairs, developed for the detection of noroviruses in stool specimens from humans or bovines .
Real-time RT-PCR system
The real-time PCR assays were carried out on the iCycler (Biorad, Nazareth, Belgium) using iScript One-step RT-PCR kits for SYBR-Green assay (Biorad, Nazareth, Belgium) and used 2 μl of extracted RNA with 25 μl of master-mix with primers at 300 nM final concentration.
The primer set used was JV12Y-JV13I . The quantity of 117,500 copies of the internal RNA control was added with each sample. The iCycler RT-PCR protocol included the following parameters: reverse transcription for 18 minutes at 48°C, 5 minutes at 95°C, followed by 45 cycles of 10 seconds at 95°C, 20 seconds at 48°C and 45 seconds at 60°C. Data were obtained during the elongation period. After the RT-PCR reaction, melting curve analysis was performed. To remove inhibition, BSA was added at a final concentration of 400 ng/μl in the RT-PCR mix or the extracted RNA was 10-fold diluted. A negative sample was added every 18 samples. All positive samples were confirmed by sequencing RT-PCR products.
Agreement of the mean melting temperature obtained with the real-time SYBR Green assay (repeatability, 35 duplicates and 10 triplicates, and reproducibility, 33 twice and 5 threefold) was measured according to a method described by Petrie and Watson .
Comparison between melting temperatures obtained with the real-time SYBR Green assay in each group (human noroviruses, bovine noroviruses and internal control) was performed using Wilcoxon rank tests and assuming unequal variance and data not distributed as a normal distribution .
All statistical analyses were carried out with Stata/SE . Relative sensitivity and specificity were estimated with 95% confidence intervals assuming a binomial exact distribution. The limit of statistical significance of the conducted tests was defined as P ≤ 0.05 and the Kappa coefficient was calculated.
This work was supported in part by SPF "Santé publique, Sécurité de la Chaîne alimentaire et Environnement" (RF6185), by Belgian Science Policy – Science for a Sustainable Development (SSD) (SD/AF/01), by the "Région Wallonne" (415701) and the University of Liège.
The authors thank Miss Pascaline Peeters for technical assistance during her practice training period and the staff of the Virology Laboratory for technical assistance. We thank especially Dr Geneviève Christiaens, Dr Patrick Goubau, Katelijne Dierick and Nadine Botteldoorn for their kind assistance and efficacy to collect samples. We thank Drs Czaplicki, Lomba (Association Régionale de Santé et d'identification animale) and De Mol (Microbiology, Faculty of Medicine, University of Liège) for their assistance in the collection of animal and human samples respectively. We are very grateful to Jennifer Cannon for her careful reading of the manuscript.
- Green KY, Chanock RM, Kapikian AZ: Human caliciviruses. In Fields Virology. Volume 1. 4th edition. Edited by: Knipe DM, Howley PM. Philadelphia: Lippincott Williams and Wilkins; 2001:841-874.
- Kapikian AZ: The discovery of the 27-nm Norwalk virus: an historic perspective. J Infect Dis 2000,181(Suppl 2):S295-S302. 10.1086/315584View ArticlePubMed
- Verbelen V, Bodeus M, Garrino MG, Scipioni A, Kabamba B, Daube G, Thiry E, Goubau P: Hospital outbreak of gastroenteritis due to Norovirus in Belgium. Acta Clin Belg 2004, 59: 30-33.View ArticlePubMed
- Vinje J, Altena SA, Koopmans MP: The incidence and genetic variability of small round-structured viruses in outbreaks of gastroenteritis in The Netherlands. J Infect Dis 1997, 176: 1374-1378.View ArticlePubMed
- Vinje J, Koopmans MP: Molecular detection and epidemiology of small round-structured viruses in outbreaks of gastroenteritis in the Netherlands. J Infect Dis 1996, 174: 610-615.View ArticlePubMed
- Green KY: The role of human caliciviruses in epidemic gastroenteritis. Arch Virol Suppl 1997, 13: 153-165.PubMed
- Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, Griffin PM, Tauxe RV: Food-related illness and death in the United States. Emerg Infect Dis 1999, 5: 607-625.PubMed CentralView ArticlePubMed
- Scipioni A, Mauroy A, Vinje J, Thiry E: Animal noroviruses. Vet J 2008, in press. doi: 10.1016/j.tvjl.2007.11.012
- Sugieda M, Nagaoka H, Kakishima Y, Ohshita T, Nakamura S, Nakajima S: Detection of Norwalk-like virus genes in the caecum contents of pigs. Arch Virol 1998, 143: 1215-1221. 10.1007/s007050050369View ArticlePubMed
- Woode GN, Bridger JC: Isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves. J Med Microbiol 1978, 11: 441-452.View ArticlePubMed
- Duizer E, Schwab KJ, Neill FH, Atmar RL, Koopmans MP, Estes MK: Laboratory efforts to cultivate noroviruses. J Gen Virol 2004, 85: 79-87. 10.1099/vir.0.19478-0View ArticlePubMed
- Straub TM, Honer zu Bentrup K, Orosz-Coghlan P, Dohnalkova A, Mayer BK, Bartholomew RA, Valdez CO, Bruckner-Lea CJ, Gerba CP, Abbaszadegan M, Nickerson CA: In vitro cell culture infectivity assay for human noroviruses. Emerg Infect Dis 2007, 13: 396-403.PubMed CentralView ArticlePubMed
- Jiang X, Wang M, Wang K, Estes MK: Sequence and genomic organization of Norwalk virus. Virol 1993, 195: 51-61. 10.1006/viro.1993.1345View Article
- Lambden PR, Caul EO, Ashley CR, Clarke IN: Sequence and genome organization of a human small round-structured (Norwalk-like) virus. Science 1993, 259: 516-519. 10.1126/science.8380940View ArticlePubMed
- Atmar RL, Estes MK: Diagnosis of noncultivatable gastroenteritis viruses, the human caliciviruses. Clin Microbiol Rev 2001, 14: 15-37. 10.1128/CMR.14.1.15-37.2001PubMed CentralView ArticlePubMed
- Vennema H, de Bruin E, Koopmans M: Rational optimization of generic primers used for Norwalk-like virus detection by reverse transcriptase polymerase chain reaction. J Clin Virol 2002, 25: 233-235. 10.1016/S1386-6532(02)00126-9View ArticlePubMed
- Kageyama T, Kojima S, Shinohara M, Uchida K, Fukushi S, Hoshino FB, Takeda N, Katayama K: Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR. J Clin Microbiol 2003, 41: 1548-1557. 10.1128/JCM.41.4.1548-1557.2003PubMed CentralView ArticlePubMed
- Menton JF, Kearney K, Morgan JG: Development of a real-time RT-PCR and Reverse Line probe Hybridisation assay for the routine detection and genotyping of Noroviruses in Ireland. Virol J 2007, 4: 86. 10.1186/1743-422X-4-86PubMed CentralView ArticlePubMed
- Nordgren J, Bucardo F, Dienus O, Svensson L, Lindgren PE: Novel light-upon-extension real-time PCR assays for detection and quantification of genogroup I and II noroviruses in clinical specimens. J Clin Microbiol 2008, 46: 164-170. 10.1128/JCM.01316-07PubMed CentralView ArticlePubMed
- Wolf S, Williamson WM, Hewitt J, Rivera-Aban M, Lin S, Ball A, Scholes P, Greening GE: Sensitive Multiplex Real-Time Reverse Transcription-PCR Assay for the Detection of Human and Animal Noroviruses in Clinical and Environmental Samples. Appl Environ Microbiol 2007, 73: 5464-5470. 10.1128/AEM.00572-07PubMed CentralView ArticlePubMed
- Scipioni A, Bourgot I, Mauroy A, Ziant D, Saegerman C, Daube G, Thiry E: Detection and quantification of human and bovine noroviruses by a TaqMan RT-PCR assay with a control for inhibition. Mol Cell Probes 2008, in press. doi: 10.1016/j.mcp.2008.02.003
- Atmar RL, Neill FH, Romalde JL, F LG, Woodley CM, Metcalf TG, Estes MK: Detection of Norwalk virus and hepatitis A virus in shellfish tissues with the PCR. Appl Environ Microbiol 1995, 61: 3014-3018.PubMed CentralPubMed
- De Leon R, Matsui SM, Baric RS, Herrmann JE, Blacklow NR, Greenberg HB, Sobsey MD: Detection of Norwalk virus in stool specimens by reverse transcriptase-polymerase chain reaction and nonradioactive oligoprobes. J Clin Microbiol 1992, 30: 3151-3157.PubMed CentralPubMed
- Le Guyader F, Estes MK, Hardy ME, Neill FH, Green J, Brown DW, Atmar RL: Evaluation of a degenerate primer for the PCR detection of human caliciviruses. Arch Virol 1996, 141: 2225-2235. 10.1007/BF01718228View ArticlePubMed
- Schwab KJ, Neill FH, Fankhauser RL, Daniels NA, Monroe SS, Bergmire-Sweat DA, Estes MK, Atmar RL: Development of methods to detect "Norwalk-like viruses" (NLVs) and hepatitis A virus in delicatessen foods: application to a food-borne NLV outbreak. Appl Environ Microbiol 2000, 66: 213-218.PubMed CentralView ArticlePubMed
- Lantz P-G, Matsson M, Wadstrom T, Radstrom P: Removal of PCR inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to PCR. J Microbiol Methods 1997, 28: 159-167. 10.1016/S0167-7012(97)00979-2View Article
- Monteiro L, Bonnemaison D, Vekris A, Petry KG, Bonnet J, Vidal R, Cabrita J, Megraud F: Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model. J Clin Microbiol 1997, 35: 995-998.PubMed CentralPubMed
- Kleiboeker SB: Applications of competitor RNA in diagnostic reverse transcription-PCR. J Clin Microbiol 2003, 41: 2055-2061. 10.1128/JCM.41.5.2055-2061.2003PubMed CentralView ArticlePubMed
- Wilson IG: Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 1997, 63: 3741-3751.PubMed CentralPubMed
- van der Poel WH, Vinje J, van der Heide R, Herrera MI, Vivo A, Koopmans MP: Norwalk-like calicivirus genes in farm animals. Emerg Infect Dis 2000, 6: 36-41.PubMed CentralView ArticlePubMed
- Helps C, Lait P, Tasker S, Harbour D: Melting curve analysis of feline calicivirus isolates detected by real-time reverse transcription PCR. J Virol Methods 2002, 106: 241-244. 10.1016/S0166-0934(02)00167-2View ArticlePubMed
- Richards GP, Watson MA, Fankhauser RL, Monroe SS: Genogroup I and II noroviruses detected in stool samples by real-time reverse transcription-PCR using highly degenerate universal primers. Appl Environ Microbiol 2004, 70: 7179-7184. 10.1128/AEM.70.12.7179-7184.2004PubMed CentralView ArticlePubMed
- Kim K, Park J, Chung Y, Cheon D, Lee IB, Lee S, Yoon J, Cho H, Song C, Lee KH: Use of internal standard RNA molecules for the RT-PCR amplification of the faeces-borne RNA viruses. J Virol Methods 2002, 104: 107-115. 10.1016/S0166-0934(02)00016-2View ArticlePubMed
- Schwab KJ, Estes MK, Neill FH, Atmar RL: Use of heat release and an internal RNA standard control in reverse transcription-PCR detection of Norwalk virus from stool samples. J Clin Microbiol 1997, 35: 511-514.PubMed CentralPubMed
- Trujillo AA, McCaustland KA, Zheng DP, Hadley LA, Vaughn G, Adams SM, Ando T, Glass RI, Monroe SS: Use of TaqMan real-time reverse transcription-PCR for rapid detection, quantification, and typing of norovirus. J Clin Microbiol 2006, 44: 1405-1412. 10.1128/JCM.44.4.1405-1412.2006PubMed CentralView ArticlePubMed
- Kreader CA: Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl Environ Microbiol 1996, 62: 1102-1106.PubMed CentralPubMed
- Cheetham S, Souza M, Meulia T, Grimes S, Han MG, Saif LJ: Pathogenesis of a genogroup II human norovirus in gnotobiotic pigs. J Virol 2006, 80: 10372-10381. 10.1128/JVI.00809-06PubMed CentralView ArticlePubMed
- Costantini V, Loisy F, Joens L, Le Guyader FS, Saif LJ: Human and animal enteric caliciviruses in oysters from different coastal regions of the United States. Appl Environ Microbiol 2006, 72: 1800-1809. 10.1128/AEM.72.3.1800-1809.2006PubMed CentralView ArticlePubMed
- Petrie A, Watson P: Additional techniques. In Statistics for veterinary and animal science. Volume 14. 2nd edition. Edited by: Science B. Edinburgh: Blackwell Science; 2006:191-211.
- Dagnelie P: Interférence statistique à une et à deux dimensions. In Statistique théorique et appliquée. Volume 2. Paris, Bruxelles: De Boeck & Larcier; 1998:659.
- StataCorp: Stata Statistical Software. In Release 8.0 edition. College Station, TX: Stata Corporation; 2003.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.