Single-tube Reverse Transcription-polymerase Chain Reaction Research Articles

First Report of Grapevine leafroll associated virus-3 in American Vitis spp. Grapevines in Washington State.

Washington State is the largest producer of juice grapes (Vitis labruscana 'Concord' and Vitis labrusca 'Niagara') and ranks second in wine grape production in the United States. Grapevine leafroll disease (GLD) is the most wide spread and economically significant virus disease in wine grapes in the state. Previous studies (2) have shown that Grapevine leafroll associated virus-3 (GLRaV-3) is the predominant virus associated with GLD. However, little is known about the incidence and economic impact of GLD on juice and table grapes. Because typical GLD symptoms may not be obvious among these cultivars, the prevalence and economic impact of GLD in Concord and Niagara, the most widely planted cultivars in Washington State, has received little attention from the grape and nursery industries. During the 2005 growing season, 32 samples from three vineyards and one nursery of 'Concord' and three samples from one nursery of 'Niagara' were collected randomly. Petiole extracts were tested by single-tube reverse transcription-polymerase chain reaction (RT-PCR; 3) with primers LC 1 (5'-CGC TAG GGC TGT GGA AGT ATT-3') and LC 2 (5'-GTT GTC CCG GGT ACC AGA TAT-3'), specific for the heat shock protein 70 homologue (Hsp70h gene) of GLRaV-3 (GenBank Accession No. AF037268). One 'Niagara' nursery sample and eleven 'Concord' samples from the three vineyards tested positive for GLRaV-3, producing a single band of the expected size of 546 bp. The 'Niagara' and six of the 'Concord' RT-PCR products were cloned in pCR2.1 (Invitrogen Corp, Carlsbad, CA) and the sequences (GenBank Accession Nos. DQ780885, DQ780886, DQ780887, DQ780888, DQ780889, DQ780890, and DQ780891) compared with the respective sequence of a New York isolate of GLRaV-3 (GenBank Accession No. AF037268). The analysis revealed that GLRaV-3 isolates from 'Concord' and 'Niagara' share nucleotide identities of 94 to 98% and amino acid identities and similarities of 97 to 98% with the Hsp70h gene homologue of the New York isolate of GLRaV-3. Additional testing by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using antibodies specific to GLRaV-3 (BIOREBA AG, Reinach, Switzerland) further confirmed these results in the 'Niagara' and two of the 'Concord' isolates. GLRaV-3 has previously been reported in labrusca cvs. Concord and Niagara in western New York (4) and Canada (1), but to our knowledge, this is the first report of GLRaV-3 in American grapevine species in the Pacific Northwest. Because wine and juice grapes are widely grown in proximity to each other in Washington State and grape mealybug (Pseudococcus maritimus), the putative vector of GLRaV-3, is present in the state vineyards, further studies will focus on the role of American grapevine species in the epidemiology of GLD.

A Single-Tube Multiplex Reverse Transcription—Polymerase Chain Reaction for Detection and Differentiation of Vesicular Stomatitis Indiana 1 and New Jersey Viruses in Insects

A multiplex single-tube reverse transcription-polymerase chain reaction (RT-PCR) has been developed for the detection and differentiation of vesicular stomatitis viruses (VSV), Indiana 1 and New Jersey, from insect samples. Using this assay, detection of either or both viruses in as little as 20 fg of total RNA from tissue culture was achieved, along with detection of vesicular stomatitis (VS) RNA from macerates containing 2 infected mosquitoes in pools of 10-30 noninfected mosquitoes. Vesicular stomatitis virus was detected by RT-PCR in all culture-positive samples, and detection as low as 4 plaque forming units per milliliter was achieved. Comparison between RT-PCR and tissue culture revealed that RT-PCR was able to detect VSV in a volume of insect macerate averaging almost 100 times less than that required for detection by tissue culture. The reported RT-PCR is a potential valuable tool for rapid and sensitive detection and differentiation of VS in insects because intense work associated with viral isolation, the cytotoxicity of insect extracts, and separate virus identification steps can be avoided. Potential application to detection and differentiation of VSV serotypes from vertebrate hosts is addressed.

Aromatase P450 messenger RNA expression in eutopic endometrium is not a specific marker for pelvic endometriosis

Objective: To determine whether expression of aromatase P450 mRNA in eutopic endometrium is predictive of the presence of pelvic endometriosis. Design: A prospective, multicenter, observational study. Setting: Four tertiary centers for reproductive medicine. Patient(s): Sixty subjects of reproductive age undergoing laparoscopy for subfertility exploration, pain assessment, or sterilization. Intervention(s): Endometrial biopsy at time of laparoscopy. Main Outcome Measure(s): The expression of aromatase P450 mRNA in endometrial specimens was determined by single-tube reverse transcription-polymerase chain reaction (RT-PCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was amplified in parallel to exclude amplification failure. Result(s): The RT-PCR amplification was successful in 56 of the 60 biopsies (93%). Pelvic endometriosis was diagnosed in 34 patients (61%) and was strongly associated with aromatase P450 mRNA expression in eutopic endometrium. As a diagnostic marker for endometriosis, aromatase P450 mRNA expression yielded a sensitivity of 82%, a specificity of 59%, a positive predictive value of 76%, and a negative predictive value of 67%. If additional uterine pathology was taken in account, the sensitivity increased to 84%, the specificity to 72%, the positive predictive value to 87%, but the negative predictive value remained unchanged (67%). Conclusion(s): Although endometrial aromatase P450 gene expression is highly predictive of the presence of pelvic disease, the relative high incidence of false-negative results and lack of specificity is likely to impair clinical application.

Preliminary report on a single-tube, non-interrupted reverse transcription-polymerase chain reaction for the detection of rabies virus in brain tissue.

A simple method for the rapid detection of rabies virus was developed employing a single-tube reverse transcription polymerase chain reaction (RT-PCR). The method utilized a single buffer system for both RT and PCR and was performed without interruption as a single thermal cycling programme. Two primer sets within the genes coding for rabies nucleoprotein and glycoprotein were used to amplify a 533 bp and a 406 bp amplicon, respectively. The amplified products were detected with a challenge virus strain (CVS) of rabies. There was no amplified product with uninfected mouse or dog brain. The method was applied to detect rabies virus in 10 mouse inoculation test (MIT)-positive and three MIT-negative brain tissue samples. The amplified product was found only in the MIT-positive samples. The amplified product was confirmed by restriction endonuclease analysis using Hinf1. The results from RT-PCR correlated well with the results from MIT. This indicates that the single-tube RT-PCR may be a useful method for detecting rabies virus in brain tissue samples from suspected cases of rabies.

The Sensitivity and Specificity of a Reverse Transcription-Polymerase Chain Reaction Assay for the Avian Pneumovirus (Colorado Strain)

A reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of avian pneumovirus (APV), Colorado strain (US/CO), was evaluated for sensitivity and specificity. The single-tube RT-PCR assay utilized primers developed from the matrix (M) gene sequence of the US/CO APV. The RT-PCR amplified the US/CO APV but did not amplify other pneumoviruses, including the avian pneumoviruses subgroups A and B. The RT-PCR was capable of detecting between 10(0.25) mean tissue culture infective dose (TCID50) and 10(-0.44) TCID50 of the US/CO APV. These results have demonstrated that the single-tube RT-PCR assay is a specific and sensitive assay for the detection of US/CO APV.

Detection of negative-stranded hepatitis C virus RNA using a novel strand-specific reverse transcription-polymerase chain reaction

We developed a novel single-tube reverse transcription-polymerase chain reaction (RT-PCR) for the specific detection of negative-stranded hepatitis C virus (HCV) RNA. By using in vitro synthesized positive- and negative-stranded HCV RNAs, it was demonstrated that as few as 50 copies of negative-stranded HCV RNA could be specifically detected with a set of primers that amplify a 232-base pair sequence unique to the 5′-non-coding region of HCV RNA, while 10 8 copies of positive-stranded HCV RNA were not detected. In addition, we demonstrated that this method allows the detection as few as 100 copies of negative-stranded HCV RNA even with the coexistence of a 100-fold excess of positive-stranded HCV RNA. Furthermore, with this method, negative-stranded HCV RNA was detected in RNAs from liver biopsy specimens obtained from patients with chronic hepatitis C, but not in RNAs from HCV-positive sera.

Intrinsic secondary structure of human TNFR-I mRNA influences the determination of gene expression by RT-PCR.

The secondary structure of human tumor necrosis factor receptor I (TNFR-I) mRNA based on its lowest folding energy was predicted. Three combinations of primers selected from open-regions and four combinations of primers from closed-regions of TNFR-I mRNA structure were employed for single-tube reverse transcription-polymerase chain reaction (RT-PCR) for the determination of TNFR-I gene expression in U937 cell. All the primers were designed with the same criteria. However, the different primers generated distinct quantities of RT-PCR products from the same concentration of TNFR-I mRNA, implying that the determination of gene expression by RT-PCR was affected by the mRNA secondary structure. In addition, the sensitivity of the open-region RT-PCR was approximately one hundred-fold higher than that in the closed-regions of TNFR-I mRNA. The low efficiency of the closed-region RT-PCR was not correlated with the G/C content of the TNFR-I mRNA structure. These results suggest that consideration of the influence of intrinsic mRNA structure of a gene is essential prior to the determination of gene expression by quantitative RT-PCR, and this open-region strategy of primer design may yield an efficient primer for in vitro amplification of cDNA by RT-PCR.