Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon.

The genetic study of RNA viruses is greatly facilitated by the availability of infectious cDNA clones. However, their construction has often been difficult. While exploring ways to simplify the construction of infectious clones, we have successfully modified and applied the newly described technique of "long PCR" to the synthesis of a full-length DNA amplicon from the RNA of a cytopathogenic mutant (HM 175/24a) of the hepatitis A virus (HAV). Primers were synthesized to match the two extremities of the HAV genome. The antisense primer, homologous to the 3' end, was used in both the reverse transcription (RT) and the PCR steps. With these primers we reproducibly obtained a full-length amplicon of approximately 7.5 kb. Further, since we engineered a T7 promoter in the sense primer, RNA could be transcribed directly from the amplicon with T7 RNA polymerase. Following transfection of cultured fetal rhesus kidney cells with the transcription mixture containing both the HAV cDNA and the transcribed RNA, replicating HAV was detected by immunofluorescence microscopy and, following passage to other cell cultures, by focus formation. The recovered virus displayed the cytopathic effect and large plaque phenotype typical of the original virus; this result highlights the fidelity of the modified long reverse transcription-PCR procedure and demonstrates the potential of this method for providing cDNAs of viral genomes and simplifying the construction of infectious clones.

Identification of early-stage colorectal cancer patients at risk of relapse post-resection by immunobead reverse transcription-PCR analysis of peritoneal lavage fluid for malignant cells

Purpose: Colorectal cancer patients diagnosed with stage I or II disease are not routinely offered adjuvant chemotherapy following resection of the primary tumor. However, up to 10% of stage I and 30% of stage II patients relapse within 5 years of surgery from recurrent or metastatic disease. The aim of this study was to determine if tumor-associated markers could detect disseminated malignant cells and so identify a subgroup of patients with early-stage colorectal cancer that were at risk of relapse. Experimental Design: We recruited consecutive patients undergoing curative resection for early-stage colorectal cancer. Immunobead reverse transcription-PCR of five tumor-associated markers (carcinoembryonic antigen, laminin γ2, ephrin B4, matrilysin, and cytokeratin 20) was used to detect the presence of colon tumor cells in peripheral blood and within the peritoneal cavity of colon cancer patients perioperatively. Clinicopathologic variables were tested for their effect on survival outcomes in univariate analyses using the Kaplan-Meier method. A multivariate Cox proportional hazards regression analysis was done to determine whether detection of tumor cells was an independent prognostic marker for disease relapse. Results: Overall, 41 of 125 (32.8%) early-stage patients were positive for disseminated tumor cells. Patients who were marker positive for disseminated cells in post-resection lavage samples showed a significantly poorer prognosis (hazard ratio, 6.2; 95% confidence interval, 1.9-19.6; P = 0.002), and this was independent of other risk factors. Conclusion: The markers used in this study identified a subgroup of early-stage patients at increased risk of relapse post-resection for primary colorectal cancer. This method may be considered as a new diagnostic tool to improve the staging and management of colorectal cancer. © 2006 American Association for Cancer Research.

Generic detection of coronaviruses and differentiation at the prototype strain level by reverse transcription-PCR and nonfluorescent low-density microarray

A nonfluorescent low-cost, low-density oligonucleotide array was designed for detecting the whole coronavirus genus after reverse transcription (RT)-PCR. The limit of detection was 15.7 copies/reaction. The clinical detection limit in patients with severe acute respiratory syndrome was 100 copies/sample. In 39 children suffering from coronavirus 229E, NL63, OC43, or HKU1, the sensitivity was equal to that of individual real-time RT-PCRs.

The inhibitory effect of pentobarbitone on reverse transcription-PCR

Pentobarbitone sodium (Sodium 5-ethyl-5[1-methylbutyl]-pentobarbitone) is a short-acting barbiturate that is commonly used to euthanase animals. As part of our studies into the molecular genetics of copper toxicosis in Bedlington terrier dogs, reverse-transcription (RT)-PCR was noted to always fail on RNA samples collected from livers of dogs sacrificed by pentobarbitone injection. When samples were collected without pentobarbitone, however, RTPCR was always successful. We suspected the possible inhibition by pentobarbitone sodium of either reverse transcriptase or Taq polymerase. In vitro studies showed that pentobarbitone interference of PCR occurred at >4 mug/mul. To identify if pentobarbitone produced competitive inhibition, each components (Taq polymerase, MgCl2, dNTP, etc.) of the PCR was individually altered. However, inhibition still persisted, suggesting that multiple PCR components may be affected. Also it was shown that pentobarbitone interference was not dependent on the PCR product size. Simple dilution of pentobarbitone contaminated DNA solutions, and the addition of bovine serum albumin (BSA) to the PCR mix overcame pentobarbitone interference. In vivo, PCR by pentobarbitone was found to be compounded by high DNA concentration and pentobarbitone contamination. In addition, both high DNA concentration and pentobarbitone contamination could be overcome through dilution and the addition of BSA. Further work is required to quantify pentobarbitone concentration in the liver-extracted DNA and RNA samples before this inhibition effect on PCR can be fully elucidated. (C) 2004 Elsevier B.V. All rights reserved.

Development of an immunomagnetic capture-reverse transcriptase-PCR assay for three pineapple ampeloviruses

A semi-automated, immunomagneticcapture-reverse transcription PCR(IMC-RT-PCR) assay for the detection of three pineapple-infecting ampeloviruses, Pineapple mealybug wilt-associated virus-1, -2 and -3, is described. The assay was equivalent in sensitivity but more rapid than conventional immunocapture RT-PCR. The assay can be used either as a one- or two-step RT-PCR and allows detection of the viruses separately or together in a triplex assay from fresh, frozen or freeze-dried pineapple leaf tissue. This IMC-RT-PCR assay could be used for high throughput screening of pineapple planting propagules and could easily be modified for the detection of other RNA viruses in a range of plant species, provided suitable antibodies are available.

Determination of toxigenic capacity by reverse transcription polymerase chain reaction in coagulase-negative staphylococci and Staphylococcus aureus isolated from newborns in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

沙冬青抗冻组份的分离纯化和afp基因克隆的尝试

本文用三种方法分离纯化沙冬青（Ammopiptanthus mongolicus）叶片抗冻蛋白质。从70%硫酸铵沉淀后的上清液中得到了一种碱性的小分子物质，UiS，具有较高的溶解度( 260 mg/ml)和较强的降低溶液冰点的能力（200 mg/ml，冰点小于-20℃）：从10%硫酸铵和26%氯化铵共沉淀物中分离到一种抗冻蛋白-B3，B3分子量为39.8 kDa，热滞活性为0.45℃(10 mg/ml)：从沙冬青叶片热稳定蛋白质中用双向电泳一电泳回收法分离到afp,其分子量为40 kDa,pl为9.0。热滞活性为0.9℃(20 mg/ml)，和其他抗冻蛋白质进行比较，没有发现相同的类型。afp和uis在叶片含量都很高，可能是沙冬青抗冻生理过程中两种主要物质，它们对于沙冬青抵御-20℃左右的冷冻温度具有重要的意义。afp N端序列为SDDL SFTF NKFV PCQT DILF，据此合成了六段5’引物和一段3’引物，用RT - PCR方法从叶片中扩增出一段200 bp左右的片段。

Selection and validation of reference genes for gene expression studies by reverse transcription quantitative PCR in Xanthomonas citri subsp citri during infection of Citrus sinensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription

Early HIV-1 reverse transcription can be separated into initiation and elongation phases. Here we show, using PCR analysis of negative-strand strong-stop DNA [(−)ssDNA] synthesis in intact virus, that different reverse transcriptase (RT) inhibitors affect distinct phases of early natural endogenous reverse transcription (NERT). The effects of nevirapine on NERT were consistent with a mechanism of action including both specific and nonspecific binding events. The nonspecific component of this inhibition targeted the elongation reaction, whereas the specific effect seemed principally to be directed at very early events (initiation or the initiation-elongation switch). In contrast, foscarnet and the nucleoside analog ddATP inhibited both early and late (−)ssDNA synthesis in a similar manner. We also examined compounds that targeted other viral proteins and found that Ro24-7429 (a Tat antagonist) and rosmarinic acid (an integrase inhibitor) also directly inhibited RT. Our results indicate that NERT can be used to identify and evaluate compounds that directly target the reverse transcription complex.

A universal test to determine the integrity of RNA, and its suitability for reverse transcription, in animal tissue laboratory specimens

Degradation of RNA in diagnostic specimens can cause false-negative test results and potential misdiagnosis when tests rely on the detection of specific RNA sequence. Current molecular methods of checking RNA integrity tend to be host species or group specific, necessitating libraries of primers and reaction conditions. The objective here was to develop a universal (multi-species) quality assurance tool for determining the integrity of RNA in animal tissues submitted to a laboratory for analyses. Ribosomal RNA (16S rRNA) transcribed from the mitochondrial 16S rDNA was used as template material for reverse transcription to cDNA and was amplified using polymerase chain reaction (PCR). As mitochondrial DNA has a high level of conservation, the primers used were shown to reverse transcribe and amplify RNA from every animal species tested. Deliberate degradation of rRNA template through temperature abuse of samples resulted in no reverse transcription and amplification. Samples spiked with viruses showed that single-stranded viral RNA and rRNA in the same sample degraded at similar rates, hence reverse transcription and PCR amplification of 16S rRNA could be used as a test of sample integrity and suitability for analysis that required the sample's RNA, including viral RNA. This test will be an invaluable quality assurance tool for determination of RNA integrity from tissue samples, thus avoiding erroneous test results that might occur if degraded target RNA is used unknowingly as template material for reverse transcription and subsequent PCR amplification.

A detection method for grass carp hemorrhagic virus (GCHV) based on a reverse transcription polymerase chain reaction

A rapid, sensitive and highly specific detection method for grass carp hemorrhagic virus (GCHV) based on a reverse transcription-polymerase chain reaction (RT-PCR) has been developed. Two pairs of PCR primers were synthesized according to the cloned cDNA sequences of the GCHV-861 strain. For each primer combination, only one specific major product was obtained when amplification was performed by using the genomic dsRNA of GCHV-861 strain. The lengths of their expected products were 320 and 223 bp, respectively. No products were obtained when nucleic acids other than GCHV-861 genomic RNA were used as RT-PCR templates. To assess the sensitivity of the method, dilutions of purified GCHV-861 dsRNA total genome (0.01 pg up to 1000 pg) were amplified and quantities of as little as 0.1 pg of purified dsRNA were detectable when the amplification product was analyzed by 1.5% agarose gel electrophoresis. This technique could detect GCHV-861 not only in infected cell culture fluids, but also in infected grass carp Ctenopharyngodon idellus and rare minnow Gobiocypris rarus with or without hemorrhagic symptoms. The results show that the RT-PCR amplification method is useful for the direct detection of GCHV.

Pan-serotypic detection of foot-and-mouth disease virus using a minor groove binder probe reverse transcription polymerase chain reaction assay

A novel assay for the pan-serotypic detection of foot-and-mouth disease virus (FMDV) was designed using a 5' conjugated minor groove binder (MGB) probe real-time RT-PCR system. This assay targets the 3D region of the FMDV genome and is capable of detecting 20 copies of a transcribed RNA standard. The linear range of the test was eight logs from 2 x 10(1) to 2 x 10(8) copies and amplification time was approximately 2 h. Using a panel of 83 RNA samples from representative FMDV isolates, the diagnostic sensitivity of this test was shown to be equivalent to a TaqMan real-time RT-PCR that targets the 5' untranslated region of FMDV. Furthermore, the assay does not detect viruses causing similar clinical diseases in pigs such as swine vesicular disease virus and vesicular stomatitis virus, nor does it detect marine caliciviruses causing vesicular exanthema. The development of this assay provides a useful tool for the differential diagnosis of FMD, potentially for use in statutory or emergency testing programmes, or for detection of FMDV RNA in research applications. (C) 2011 Elsevier B.V. All rights reserved.

RT-Bst: an integrated approach for reverse transcription and enrichment of cDNA from viral RNA

The synthesis of cDNA from RNA is challenging due to the inefficiency of reverse transcription (RT). In order to address this, a method was developed known as RT-Bst for sequential RT of RNA and Bst DNA polymerase amplification for enrichment of cDNA in a single tube reaction. Using genomic RNA from bacteriophage MS2, the yield of cDNA produced by RT alone and RT-Bst were compared by analysis of PCR-amplified products. Using random primers a superior performance was observed when amplifying MS2 RNA following RT-Bst compared to RT alone, indicating that greater quantities of cDNA were present after RT-Bst. RT-Bst was also compared with RT alone for their relative ability to produce sufficient cDNA to amplify 8 target regions spanning the respiratory syncytial virus (RSV) genome. Six out of 8 targets were amplified consistently by PCR subsequent to RT-Bst amplification whereas only 3 out of 8 targets could be amplified after RT alone. RSV sequences were selectively amplified using RSV specific primers from a mixed template containing an excess of MS2 RNA in a RT-Bst reaction without amplifying MS2 sequences. This suggests that RT-Bst can be used to amplify RNA sequences non-specifically using random primers and specifically using sequence specific primers and enhances the yield of cDNA when compared to RT alone.