Sensitive multiplex real-time PCR and RT-PCR assays with CICs were established to detect XMRV proviral DNA in PBMCs or viral RNA in plasma, respectively, from Chinese patients with CFS. The virus was not detected in any of our study subjects; these results do not support an association between XMRV and CFS in Chinese.
Our findings appeared to be inconsistent with a previous report of XMRV DNA isolation from PBMCs of CFS patients in the US [3
]. Technical differences can be ruled out as a reason for the failure to detect XMRV. The sensitivity of the multiplex real-time PCR (20 copies/reaction) was likely to be as sensitive, if not more so, as the end point PCR method previously used [3
], thus suggesting that multiplex real-time PCR can be used for the detection of XMRV proviral DNA. The end-point PCR method used in the previous study requires multiple manipulations of the sample after the amplification step, thus increasing the risk of carryover contamination. The possibility that proviral DNA degradation during the extraction process may have led to our negative results seems unlikely. The β-actin gene was positive for all clinical specimens, confirming the integrity of the DNA. In addition, samples used in this research were representative of typical patients with CFS, which met the 1994 Centers for Disease Control and Prevention case definition of CFS (called the Fukuda criteria). Although the patients studied by Lombardi et al [3
] were reported to fulfil the same criteria, a clear description of their patient and control cohorts was lacking.
Several PCR-based methodologies have been developed for the detection of XMRV DNA, including end point and real-time PCR methodologies [4
]. Beta-globin gene, GAPDH, and β-actin were used as non-competitive ICs to validate DNA integrity in 4 recent studies of XMRV [4
]. However, DNA concentrations may vary widely between clinical samples. Van Kuppeveld et al [6
] amplified a known amount of phocine distemper virus (PDV) that had been added to clinical samples to monitor RNA quality and to detect amplification inhibition. Although attractive, the use of live viruses as internal controls may raise concerns regarding safety and consistency between preparations. Additionally, the performance of non-competitive ICs is imperfect due to differences in the amplification efficiencies of different target nucleic acids [15
]. Here, we used CIC as a substitute. The CIC was a constructed plasmid which mimicked the template with the same length and primer binding sites, and similar GC content. In order to avoid suppression of target amplification and possible competition between the target and CIC, we optimized the concentration of CIC added to the samples. IC concentrations of more than 1000 copies/μl altered the Ct of almost all standards which yielded an underestimation of the concentration. CIC at 1000 copies/μl was determined to be optimal for our real-time assay. Our results indicated that no inhibitory effects were at play in the multiplex real-time assay we used to screen our study population.
The pathogenesis and outcome of XMRV infection may be associated or even causally linked with plasma viral RNA loads, as well as proviral loads. In addition to XMRV proviral DNA detection, we developed a sensitive multiplex real-time RT-PCR assay to detect XMRV viral RNA in plasma. We constructed armored RNAs to serve as the XMRV viral RNA standard and CIC to evaluate the analytical linearity, sensitivity, specificity, and reproducibility of the detection assay. Both were stable in normal human EDTA-preserved plasma at 4°C, 37°C, and room temperature for over 3 months. Armored RNA is a kind of non-infectious recombinant virus-like particle (VLP) containing target exogenous RNA. In comparison to naked RNA, as armored RNA is a more suitable candidate for a positive control or standard in the quantification of RNA viruses, because it is RNase-resistant, stable, non-infectious, and easily extracted by conventional methods [16
]. Moreover, armored RNA can serve as a control for all stages of the RT-PCR assay, from extraction through amplification. The inclusion of the HCV 5'UTR made it easy to assign an IU value to the XMRV RNA and the CIC within the armored RNA, avoiding the necessity for the complex procedures involved in value assignment of calibrators or standards when international standards (IS) are not available [12
]. These characteristics of armored RNA ensure the validity of our data. Conflicting results have made the associations between XMRV and CFS unclear; it is therefore important to produce a 'universal' XMRV standard so that the results of different assays may be compared. By using an armored RNA standard, different research groups and clinical laboratories may directly compare their quantitative data. Nevertheless, we did not detect XMRV viral RNA with our armored RNA-standardised method in plasma samples from a Chinese study population.
These findings may not be generalisable beyond the study population because XMRV infection rates may vary geographically. Similarly, although XMRV was initially discovered in tumour tissues of a subset of patients with prostate cancer [20
], other studies have shown a variable incidence of the virus in prostate tumours. One US study found XMRV in up to 23% of prostate cancer tumours [21
]; however, a recent German study found a 0% incidence of XMRV [22
]. Additional research is needed to determine what, if any, role XMRV plays in CFS in Chinese patients.