A TaqMan real-time RT-PCR for quantifying Mourilyan virus infection levels in penaeid shrimp tissues

A highly sensitive and specific TaqMan real-time quantitative RT-PCR (qRT-PCR) was developed to detect and quantify Mourilyan virus (MoV), a newly described bunya-like virus of penaeid shrimp. The PCR primers and TaqMan probe targeted a 67-nucleotide (nt) sequence in the MoV M RNA segment. Using dilution series of a 849 nt RNA transcribed in vitro from cDNA clone pMoV4.1, the assay could detect down to a single MoV RNA equivalent, reliably detected 10 RNA copies and had a log linear range up to 1 x 10(9) RNA copies. In experimentally infected Penaeus japonicus shrimp, the test was used to quantify increases in MoV loads over time in hemocytes, lymphoid organ and gills. Sequential increases in MoV RNA copy numbers occurred in lymphoid organ and gill tissues collected at 6, 24 and 48 h post-infection. However, RNA copy numbers decreased slightly in hemocytes sampled at 48 h compared to 24 h. The qRT-PCR data correlated well with amplicon yields generated using a conventional RT-nested PCR targeting the same MoV RNA segment. Moreover, histology and in situ hybridisation using shrimp cephalothorax sections identified increases in lymphoid organ spheroid numbers and confirmed that increases in MoV RNA detected in lymphoid organ tissue were due to expansion in the numbers of infected cells. The qRT-PCR assay should find use in high-throughput screening applications to detect MoV in broodstock and postlarvae used for culture or breeding purposes and for tracking changes in infection levels during shrimp grow-out.     

Development of a real-time multiplex PCR assay for detection of viral pathogens of penaeid shrimp

A real-time multiplex polymerase chain reaction (rtm-PCR) assay was developed and optimized to simultaneously detect three viral pathogens of shrimp in one reaction. Three sets of specific oligonucleotide primers for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV) and Taura syndrome virus (TSV), along with three TaqMan probes specific for each virus were used in the assay. The rtm-PCR results were detected and analyzed using the Light Cycler 2.0 system. Forty-five PCR-positive samples and four negative samples were used to confirm the sensitivity and specificity of the rtm-PCR. The rtm-PCR identified and differentiated the three pathogens. With one viral infection of shrimp, a specific amplified standard curve was displayed. When samples from shrimp infected with two or three pathogens were analyzed, two or three specific standard curves were displayed. The sensitivity of the rtm-PCR assay was 2,000, 20, and 2,000 template copies for WSSV, IHHNV and TSV, respectively. No positive results (standard curves) were displayed when nucleic acid from Vibro spp., and Streptococcus spp. DNA were used as PCR templates. The results indicate that real-time multiplex PCR is able to detect the presence of and differentiate each pathogen in infected shrimp. This real-time multiplex PCR assay is a quick, sensitive, and specific test for detection of WSSV, IHHNV and TSV and will be useful for the control of these viruses in shrimp.     

Quantitation of infectious myonecrosis virus in different tissues of naturally infected Pacific white shrimp, Litopenaeus vannamei, using real-time PCR with SYBR Green chemistry

The Pacific white shrimp, Litopenaeus vannamei, is the most important shrimp species in volume in world aquaculture. However, in recent decades, outbreaks of diseases, especially viral diseases, have led to significant economic losses, threatening the sustainability of shrimp farming worldwide. In 2004, Brazilian shrimp farming was seriously affected by a new disease caused by the Infectious myonecrosis virus (IMNV). Thus, disease control based on rapid and sensitive pathogen detection methods has become a priority. In this study, a specific quantitation method for IMNV was developed using real-time PCR with SYBR Green chemistry and viral load of the principal target tissues of chronically infected animals was quantified. The quantitative analysis revealed that mean viral load ranged from 5.08 × 10⁸ to 1.33 × 10⁶ copies/μg of total RNA in the hemolymph, 5.096 × 10⁵ to 1.26 × 10³ copies/μg in the pleopods, 6.85 × 10⁸ to 3.09 × 10⁴ copies/μg in muscle and 8.15 × 10⁶ to 3.90 × 10³ copies/μg in gills. Different viral loads of IMNV were found with greater values in the hemolymph and muscle, followed by the pleopods and gills.     

Real-time PCR and its application to mumps rapid diagnosis

A real-time polymerase chain reaction assay was initially developed in China to detect mumps genome. The primers and TaqMan-MGB probe were selected from regions of the hemagglutinin gene of mumps virus. The primers and probe for the real-time PCR were evaluated by both laboratories in China and in the UK using three different pieces of equipment, LightCycler (Roche), MJ DNA Engine Option 2 (BIO-RAD) and TaqMan (ABI Prism) on different samples. The reaction was performed with either a one-step (China) or two-step (UK) process. The sensitivity (10 copies) was estimated using a serial dilution of constructed mumps-plasmid DNA and a linear standard curve was obtained between 10 and 10(7) DNA copies/reaction, which can be used to quantify viral loads. The detection limit on cell culture-grown virus was approximately 2 pfu/ml with a two-step assay on TaqMan, which was equivalent to the sensitivity of the nested PCR routinely used in the UK. The specificity was proved by testing a range of respiratory viruses and several genotypes of mumps strains. The concentration of primers and probe is 22 pmol and 6.25 or 7 pmol respectively for a 25 microl reaction. The assay took 3 hr from viral RNA extraction to complete the detection using any of the three pieces of equipment. Three hundred forty-one (35 in China and 306 in the UK) clinical specimens were tested, the results showing that this real-time PCR assay is suitable for rapid and accurate detection of mumps virus RNA in various types of clinical specimens.     

Highly sensitive TaqMan RT-PCR assay for detection and quantification of both lineages of West Nile virus RNA.

BACKGROUND: Starting in 1999, the West Nile virus (WNV) epidemic represents the largest outbreak of arboviral encephalitis ever recorded in the U.S. The effective means to determine an infection are detection of viral nucleic acid and/or viral specific immunoglobulin, IgM and/or IgG. OBJECTIVE: To develop a highly sensitive and specific TaqMan RT-PCR assay for the detection and quantification of WNV RNA of lineage 1 and lineage 2. STUDY DESIGN: A TaqMan RT-PCR primer-probe was designed to perfectly match target sequences of all sequenced WNV strains and isolates, which added a layer of protection against false-negative results due to strain variability. In addition, the inclusion of a low level RNA internal control (IC) in the assay increased the precision and accuracy of the assay. RESULTS: By optimizing the RNA preparation procedure for increased WNV RNA recovery, together with optimizing the primer-probe and TaqMan conditions for improved amplification efficiency, we developed a highly sensitive assay with the detection limit of 10 copies/mL. To evaluate the assay, we tested plasma samples from 12 transfusion-transmitted implicated cases in 2002 and from 68 positive blood donors in 2003. All tested specimens were WNV positive. The viral load of 68 positive blood donor samples collected in 2003 ranged from 10 copies/mL to 67,000 copies/mL with a mean of 8100 copies/mL. Furthermore, high sensitivity of the assay was achieved without compromising specificity. All 100 routine donor samples tested negative. CONCLUSIONS: The assay results demonstrate that our in-house TaqMan RT-PCR procedure can detect and quantify WNV RNA of lineage1 and lineage 2 in human plasma with high sensitivity and specificity.     

The use of differential display to isolate viral genomic sequence for rapid development of PCR-based detection methods. A test case using Taura syndrome virus

The purpose of this study was to explore the efficacy of using differential display (DD) to isolate viral genomic sequence using tissues from infected organisms so that a PCR procedure to detect the pathogen may be developed rapidly. The model virus used was the Taura syndrome virus (TSV), a ssRNA virus that cause high rates of mortality at shrimp farms. Two random primers in combination with four anchored primers were used to isolate five cDNAs, ranging in size from 241 to 822 bp, that were differentially expressed in TSV-infected shrimp (Litopenaeus vannamei). PCR experiments revealed that four of the five encoded shrimp genes while the fifth was likely to be a TSV gene. Evidence that the putative TSV sequence is part of the TSV genome was obtained by the 97% sequence identity it shared with the published TSV genome. PCR primers were designed successfully using the differential display sequence to develop a RT-PCR-based method to detect TSV. Because differential display does not require physical isolation of the virus and only a small amount of infected sample is needed, the technique may be useful as a method to isolate nucleic acid sequences from emerging pathogens so that PCR primers for their detection may be developed rapidly.     

Quantitation of HIV-1 RNA levels in plasma and CSF of asymptomatic HIV-1 infected patients from South India using a TaqMan real time PCR assay.

BACKGROUND: Most of the quantitation assays for HIV-1 RNA used currently are designed and optimized for HIV-1 subtype B viruses and hence may not be suitable for India, where the predominant subtype is HIV-1 subtype C. OBJECTIVES: Development and standardization of HIV-1 TaqMan real time PCR assay suitable for measuring plasma and CSF viral RNA levels in HIV subtype C infected individuals. STUDY DESIGN: A TaqMan real time PCR was developed using primers and probes selected in the gag region for detection of Indian HIV-1 subtype C strain. Plasma (n=120) and CSF samples (n=46) obtained from HIV infected subjects were used to evaluate the sensitivity and specificity of the assay. A comparative evaluation was carried out with a commercially available quantitative HIV viral load assay (Roche Amplicor Version 1.5). RESULTS: The TaqMan assay was able to amplify all HIV-1 group M subtypes except subtype E. Viral loads could be estimated in all the plasma (n=120) and 40/46 CSF samples obtained from HIV positive subjects. Sensitivity of this assay was found to be 180 copies/ml. Correlation with the commercially available viral load assay was very good (r=0.885). CONCLUSIONS: A TaqMan real time PCR was standardized for HIV-1 subtype C and it was more sensitive (180 copies/ml) than standard Amplicor monitor assay, Version 1.5 (400 copies/ml).     

A one-step real time RT-PCR assay for quantifying rice stripe virus in rice and in the small brown planthopper (Laodelphax striatellus Fallen)

Rice stripe virus (RSV) is an important pathogen affecting rice production in subtropical and temperate regions. One-step real time RT-PCR methods using the TaqMan probe are described for quantitative detection of RSV in rice tissues and in Laodelphax striatellus Fallen, the small brown planthopper (SBPH). Primers and probe for specific detection of RSV were designed within the conserved region identified within the coat protein (CP) gene sequence. A DNA fragment was amplified for mimicking the complementary RNA by PCR-based gene assembly, and was used for generation of standard RNA templates. A sensitivity assay showed that the detection limit of the assay was 20 copies, and the standard curve had a linear range from 20 to 2 x 10(5) copies, with good reproducibility. An internal control assay designed to target the rice ubiquitin 5 gene (UBQ5) was included in detecting RSV in different infected rice tissues. Simultaneously, a real time RT-PCR assay was used to detect the RSV CP gene in viruliferous SBPH. The results showed that the numbers of RSV CP genes in different tissues were variable. Accumulation of the RSV CP gene was greater in rice leaf and SBPH thoraco-abdominal tissue than in rice stem and SBPH head, respectively. As determined by an end-point dilution comparison, one-step real time RT-PCR was close to 10(4)-fold more sensitive than the indirect enzyme-linked immunosorbent assay (ELISA) for RSV detection. This quantitative detection assay provides a valuable tool for diagnosis and molecular studies of RSV biology.     

Comparison of TaqMan real-time PCR and p24 Elisa for quantification of in vitro HIV-1 replication

In this study, TaqMan PCR was used to assess viral replication of HIV-1 infected cells in vitro. This PCR technique was compared with p24 ELISA as a standard method to monitor HIV-1 replication in cell culture. Hut78 T-lymphoblastoid cells were infected with different titres of HIV-1(IIIb) (MOI 0.05-0.0005). The course of HIV-1 replication was monitored by determination of p24 concentrations by ELISA in cell culture supernatants and by quantitation of HIV-1 gag RNA by TaqMan RT-PCR. Additionally, the number of HIV-1 proviral copies was assessed by TaqMan PCR. Monitoring of HIV-1 replication by p24 ELISA and TaqMan RT-PCR revealed comparable kinetics of infection. Both methods provided similar data on the exponential increase and on plateauing of HIV-1 replication. Furthermore, both methods were equally sensitive. However, a 7 log linearity of TaqMan HIV-1 gag PCR was demonstrated without dilution of the specimen, in contrast to p24 ELISA, where because of its narrow range of detectable p24 concentrations, sample dilution was necessary. Although determination of the number of proviral copies by TaqMan PCR does not measure HIV-1 replication, the kinetics of proviral copy number following in vitro inoculation of cells with HIV-1 was nearly the same as the kinetics of HIV-1 RNA copy numbers. In conclusion, TaqMan real-time RT-PCR was demonstrated as a reliable and sensitive tool to quantify and monitor HIV-1 replication in cell culture. It is suggested, therefore, that this technique be an alternative method to monitor HIV-1 replication in vitro.