Simultaneous detection of HCV and HIV-1 by SYBR Green real time multiplex RT-PCR technique in plasma samples
Introduction
HIV-1 and HCV co-infection is a relatively common clinical occurrence in Europe and in USA affecting approximately 25% of HIV-1–infected patients and 10% of HCV-infected individuals, respectively [1], [2], [3]. This high prevalence of HIV-1/HCV co-infection is closely related to common transmission routes such as blood and blood products. In particular, the prevalence of HCV infection in HIV-1 positive patients is higher in parental transmission by injecting drug use (about 90%) and by transfusion of blood or blood products (70%), whereas it is relatively lower after sexual or vertical transmission [4], [5], [6]. Although HAART treatment of HIV-1 positive patients has clearly decreased the incidence of mortality and the rate of opportunistic infection, the clinical importance of HIV-1/HCV co-infection is increasing because HCV-related liver disease has emerged as a major cause of morbidity and mortality in HAART-treated HIV-1–infected patients [7], [8]. Indeed, HCV infection is associated with an increased risk of developing hepatotoxicity in response to HAART and it has negative effects on HIV disease progression [9], [10]. On the other hand, HIV-1 co-infection accelerates HCV-related liver diseases, inducing a more rapid evolution to cyrrosis and hepatocarcinoma in these patients [11], [12]. Besides the direct clinical impact of HIV-1 and HCV co-infection, HCV and HIV-1 are two of the most important infectious agents transmitted by blood transfusion and so, reliable and sensitive screening of blood units through serological and molecular procedures is essential [13]. Although sensitive serological tests were developed successfully, a residual risk of viral infection persists, since the HIV or HCV infection may be transmitted either during the so-called window period, when viremia precedes seroconversion, or in the presence of immunovariant virus infection, where the serological tests may be ineffective [13]. To minimize this possibility, several nucleic acid tests (NAT) have been developed with a dramatically improved analytical sensitivity in comparison with direct antigen detection or virus isolation [13], [14]. However, NAT technology has a major drawback of high cost and consequent poor cost-effectiveness. Moreover, the pooling of samples employed by several NAT approaches determines a relatively high rate of false-positive results, a loss of sensitivity when a low-positive sample is diluted into the pool and the need to recognize infected unit in the pool with a delay of blood availability particularly severe for some cellular products such as platelets [13], [14]. To overcome these weaknesses, previous studies developed sensitive and rapid multiplex PCR assays that can detect the genomes of different viruses simultaneously [15], [16], [17]. In particular, some multiplex real-time PCR, approaches were developed employing TaqMan technology for viral co-infection detection [18], [19]. Recently, multiplex real-time PCR, with melting curve analysis, has been described as a simple, reliable, and rapid assay for the detection and identification of certain bacteria, protozoa, and viruses [20], [21], [22], [23], [24], [25].
This paper describes a RT-PCR multiplex real-time technique based on SYBR Green as fluorochrome for simultaneous detection of HIV-1 and HCV genomes in clinical plasma samples.
Section snippets
Patients
Thirty HIV-1/HCV co-infected patients and 15 HIV-1/HCV seronegative blood donors were enrolled in our study after informed consent, following the Helsinki declaration. HIV-1/HCV seropositive and healthy blood donor plasma samples were obtained from patients living in Italy. Sequence analysis [26] indicated that HIV-1 seropositive patients were infected by HIV-1 subtype B, whereas HCV genotype analysis of HCV-infected patients showed different genotypes such as 1a, 1b, 3a, and 4 HCV genotypes.
Branched DNA HIV-1 and HCV RNA viral load determination
SYBR Green-based real time multiplex RT-PCR optimization to detect HCV and HIV-1 genomes
We assessed a SYBR Green-based real time multiplex RT-PCR for the simultaneous detection of HIV-1 and HCV viral load in plasma samples. We selected HIV-1 and HCV specific oligonucleotides pairs able to detect all significant HIV-1 subtypes and HCV genotypes. We chose two classical HIV-1 specific oligonucleotides, represented by SK 431 and SK 462, that recognize a well-conserved region in the HIV-1 gag gene amplifying a 142-bp fragment. This pair of oligos is known to be effective in the
Discussion and conclusions
This report describes the development of a qualitative SYBR Green-based multiplex real time RT-PCR for the simultaneous detection of HCV and HIV-1 RNA in plasma samples. This multiplex RT-PCR format was assessed using an HIV-1 and HCV-specific oligonucleotide pair that encompassed RNA sequences in viral conserved genome regions in order to yield primers able to detect all the HCV genotypes and HIV-1 major subtypes. Moreover, the optimization of this assay focused initially on the compatibility
Acknowledgements
This work was supported by ‘AIDS project’ of the Italian Ministry of Health and Funds for Selected Research Topics of the University of Bologna.
References (38)
- et al.
Mother-to-child transmission of hepatitis C virus: evidence for preventable peripartum transmission
Lancet
(2000) - et al.
Hepatitis B or hepatitis C and human immunodeficiency virus infection
J Hepatol
(2005) - et al.
Clinical progression, survival, and immune recovery during antiretroviral therapy in patients with HIV-1 and hepatitis C virus coinfection: the Swiss HIV cohort study
Lancet
(2000) - et al.
Hepatocellular carcinoma in HIV-infected patients with chronic hepatitis C
Am J Gastroenterol
(2001) - et al.
High throughput screening of 16 millions serologically negative blood donors for hepatitis B virus, hepatitis C virus and human immunodeficiency virus type 1 by nucleic acid amplification testing with specific and sensitive multiplex reagent in Japan
J Virol Methods
(2003) - et al.
Multiplex real-time quantitative RT-PCR assay for hepatitis B virus, hepatitis C virus and human immunodeficiency virus type 1
J Virol Methods
(2004) - et al.
Qualitative multiplex RT-PCR for simultaneous detection of hepatitis C virus and human immunodeficiency virus in plasma samples
Clin Microbiol Infect
(2004) - et al.
Detection and differentiation of Plum pox virus using real time multiplex PCR with SYBR Green and melting curve analysis: a rapid method for strain typing
J Virol Methods
(2005) - et al.
Rapid differentiation of Old World Leishmania species by LightCycler polymerase chain reaction and melting curve analysis
J Microbiol Methods
(2002) Simultaneous detection of enteric viruses by multiplex real time RT-PCR
J Virol Methods
(2004)
A SYBR Green real-time RT-PCR method to detect and quantitate Norwalk virus in stools
J Virol Methods
Improvement in the specificity and sensitivity of detection for the Taura syndrome virus and Yellow head virus of penaeid shrimp by increasing the amplicon size
J Virol Methods
Quantitation of HCV RNA using real-time PCR and fluorimetry
J Virol Methods
SYBR green I DNA staining increases the detection sensitivity of viruses by polymerase chain reaction
J Virol Methods
The natural history of community-acquired hepatitis C in the United States. The sentinel counties chronic non-A, non-B hepatitis study team
N Engl J Med
The prevalence of hepatitis C virus infection in the United States, 1988 through 1994
N Engl J Med
Hepatitis C virus prevalence among patients infected with human immunodeficiency virus: a cross-sectional analysis of the US adult AIDS Clinical Trials Group
Clin Infect Dis
Prevalence and determinants of antibodies to hepatitis C virus and markers for hepatitis B virus infection in patients with HIV infection in Aquitaine. Groupe d'Epidemiologie Clinique du SIDA en Aquitaine
BMJ
Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV outpatient study investigators
N Engl J Med
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The devolopment of duplex real-time PCR based on SYBR Green florescence for rapid identification of ruminant and poultry origins in foodstuff
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