Development of a real‐time TaqMan PCR assay for the detection of porcine and bovine Torque teno virus

Aims: The goal of this study was to develop and to optimize molecular tools to detect the presence of Torque teno virus (TTV) in swine and cattle. A novel real‐time polymerase chain reaction (PCR) using a TaqMan probe was developed to detect both genogroups of TTV strains. Methods and Results: Oligonucleotide primers and hybridization probes were designed based on sequence analysis of the noncoding region, a highly conserved part of the genome. The real‐time PCR assay specifically detected bovine and porcine TTV DNA without cross‐amplification of other common pathogens. The assay was compared with conventional PCR and nested‐PCR assays for the detection of porcine genogroups 1 and 2 and bovine TTV on plasma and faecal samples, and the assay was found faster, more reliable and reduced the risk of false positive results. Conclusions: The real‐time PCR assay provided better detection results for the two TTV genogroups in both swine and cattle compared to the conventional PCR assays. Significance and Impact of the Study: This new TaqMan PCR assay will be a useful tool for the detection of animal TTV strains, to evaluate the viral load from animal host and finally to identify the presence of these viruses in the agri‐food continuum.     

Association of zoonotic Ljungan virus with intrauterine fetal deaths

BACKGROUND: It has recently been shown that Ljungan virus (LV) is associated with disease in its wild rodent reservoir. In addition, it has been demonstrated that LV causes malformations and perinatal death in a mouse model. The question was therefore raised whether LV is a zoonotic agent in humans. METHODS: Population fluctuations of native rodents in Sweden were compared to the incidence of intrauterine fetal deaths (IUFDs) using the Swedish national hospitalization database. Formalin-fixed tissues from cases of IUFD were investigated using LV-specific immunohistochemistry. RESULTS: Variation in the incidence of IUFDs closely tracked the fluctuations in native rodent populations. LV was detected in the brain tissue in 4 of 10 cases of IUFDs investigated by immunochemistry. LV was also detected in the placenta in 5 of the 10 IUFD cases, but in none of 20 placentas from normal pregnancies. CONCLUSIONS: LV may play an important role in IUFDs.     

Detection of Tobacco mosaic virus and Tomato mosaic virus in pepper and tomato by multiplex RT-PCR

Aims: To develop a highly sensitive and rapid protocol for simultaneous detection and differentiation of Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) in pepper and tomato. In this study, we use the multiplex PCR technique to detect dual infection of these two viruses. Methods and Results: A multiplex RT–PCR method consisting of one‐tube reaction with two primer pairs targeted to replicase genes was developed to simultaneously detect TMV and ToMV in seed samples of pepper and tomato. Specific primers were designed from conserved regions of each of the virus genomes, and their specificity was confirmed by sequencing PCR products. RT–PCR detected up to 10^?6 dilution of total RNA extracted from infected leaves. Multiplex RT–PCR revealed the presence of both TMV and ToMV in three of 18 seed samples of tomato and one of 18 seed samples of pepper. Conclusions: The multiplex PCR assay was a cost effective, quick diagnostic technique, which was helpful in differentiating TMV and ToMV accurately. Significance and Impact of the Study: The multiplex PCR assay described in this study is a valuable tool for plant pathology and basic research studies. This method may facilitate better recognition and distinction of TMV and ToMV in both pepper and tomato.     

Identification of germinal disk region derived genes potentially involved in hen fertility

Despite the regular decrease in fertility observed in hens, especially in “meat” lines, little is known about genes affecting fertility. We used the Affymetrix microarray to search for oocyte genes whose expression would vary in relation to fertility rate in both “laying” and “meat” line hens. We focused on oocyte genes because several of them have been found to be involved in fertility in other species. Based on microarray analysis, 54 and 84 genes were differentially expressed between germinal disc regions (GDR) of F1 maturation stage oocytes from hens exhibiting either high (100%) or low (from 22% to 80%) fertility rate from laying and meat lines respectively. Most of these differentially expressed genes were distributed between “laying” and “meat” lines indicating that mechanisms involved in the decrease in fertility rates in these two cases were independent. Real time RT‐PCR performed on the same samples which were used for microarray confirmed in several cases differences in gene expression levels detected by microarray. Moreover the correlations between gene expression levels and fertility rates were evaluated for the 10 most interesting genes at different stages of follicular maturation and early embryo development on individual GDR samples from hens exhibiting different fertility rates. In total, we identified five genes whose expression levels correlated with fertility rate in accordance with findings of microarray analysis and real time RT‐PCR: VWC2, CR407412, TAPA, FGL2, and TRAP6. The biological significance of these genes sheds light on potential mechanisms influencing fertility and could provide candidates for fertility markers in the hen. Mol. Reprod. Dev. 76: 1043–1055, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of chronic intermittent hypoxia on biological behavior and hypoxia‐associated gene expression in lung cancer cells

Hypoxia plays an important role in the development of solid tumors and is associated with their therapeutic resistance. There exist three major forms of hypoxia: acute, chronic, and intermittent hypoxia. Previous studies have shown that cancer cells could behave in the form of adaptation to hypoxia in tumor growth, which could result in their biological changes and determine their responses to the therapies. To investigate the tumor cells' adaptation to hypoxia, we recreated two models using two lung cancer cell lines in the presence of intermittent hypoxia, which is characterized by changes in oxygen pressure within the disorganized vascular network. We investigated biological behaviors such as cell cycle, proliferation, radiation sensitivity, apoptosis and migration, hypoxia signal pathway in the lung cancer cells treated with chronic intermittent hypoxia, as well as the role of hypoxia inducible factor 1 there, hypoxia‐inducible genes analyzed by real‐time RT‐PCR chip in H446 cells treated with the model. The results indicated the changes of some hypoxia target gene expressions of those induced by hypoxia, some of which were confirmed by real‐time RT‐PCR. The cells mediated by irradiation induced resistance to radiation and apoptosis and increased metastasis in lung cancer cells. It was found that such changes were related to hypoxia inducible factor 1, alpha subunit (HIF‐1α). J. Cell. Biochem. 111: 554–563, 2010. © 2010 Wiley‐Liss, Inc.     

Detection of adeno- and lentiviral (HIV1) contaminations on laboratory surfaces as a tool for the surveillance of biosafety standards

Aims: As a biosafety laboratory, we survey the handling of adenovirus type 5 (Ad5) and HIV1‐derived lentivirus in contained‐use facilities in Switzerland to identify insufficiencies of the safety precautions taken by the laboratories. Methods and Results: In the past 9 years, we took 430 swab samples from various types of surfaces in research laboratories. Samples were examined for Ad5 contaminations by real‐time PCR and infectivity assay or for the presence of lentivirus (HIV1) nucleic acids by real‐time (RT) PCR. Samples collected from centrifuges did not only contain Ad5 DNA more frequently but also exhibited higher numbers of Ad5 and lentiviral (HIV1) DNA copies than swabs from any other area of sampling. Five of ten samples containing infectious Ad5 particles or lentivirus (HIV1) RNA were found in samples taken from centrifuges. Ad5 contamination rates were higher in the tube holder and lower on the inner wall of the rotor chamber in centrifuges that were fitted with aerosol tight covers compared to centrifuges without covers. Conclusions: Our results allowed the comparison of hygiene standards of different laboratories and lead to the identification of centrifuges as hotspots for contaminations. Significance and Impact of the Study: Based on our results, we propose to use the collected data as a tool for rating future swab results. Furthermore, the amount of Ad5 and HIV1‐derived lentivirus DNA could serve as an indicator of the level of good laboratory practice in contained‐use laboratories handling these viral vectors.     

Development of reverse transcription loop‐mediated isothermal amplification assay for sensitive and rapid detection of Hosta virus X

The one‐step real‐time turbidity loop‐mediated isothermal amplification assay (RealAmp) was developed to detect Hosta virus X (HVX), the most devastating threat to hosta industry. The reaction was performed in a single tube at 63°C for 15 min, and real‐time turbidimetry was used to monitor the amplification results. Specificity and sensitivity analyses demonstrated that this RealAmp method was sensitive as real‐time TaqMan RT‐PCR and about 100‐fold higher than conventional RT‐PCR with no cross‐reaction with other viral pathogens. Field samples detection showed that HVX could be identified effectively with this method. Overall, this RealAmp assay for HVX detection was simple, specific, sensitive, convenient and time‐saving and could assist in the quarantine measures for prevention and control of the disease caused by HVX.     

Establishment of an efficient multiplex real‐time PCR assay for human papillomavirus genotyping in cervical cytology specimens: comparison with hybrid capture II

J.‐H. Lee, N.‐W. Lee, S.‐W. Hong, Y.‐S. Nam, J.‐W. Choi and Y.‐S. Kim Establishment of an efficient multiplex real‐time PCR assay for human papillomavirus genotyping in cervical cytology specimens: comparison with hybrid capture II Objective: To establish an efficient multiplex real‐time PCR assay for 15 human papillomavirus (HPV) genotypes, we designed multiplexing parameters and compared our PCR system with the hybrid capture (HC) II test using cervical cytology samples. Methods: For preventing cross‐reactive amplifications, variable HPV genes (E1, E2, E6, E7 and L1) were targeted. The melting temperatures of all primers and probes, and the size of the PCR product were optimized for the multiplex PCR. Our PCR system was compared with the HC II assays in the detection and genotyping of HPV infection using 173 cytology smears. Discordant cases between the two assays were verified by direct HPV DNA sequencing. Results: Of 173 women, 93 (53.8%) were HPV‐positive by the HC II assay and/or the multiplex real‐time PCR assay. The HPV genotypes were determined in 92 (98.9%) of 93 cases by the multiplex real‐time PCR and/or DNA sequencing. The agreement rate between multiplex PCR and HC II methods was 91.9% (kappa = 0.84). Although the sample size of this study needs to be increased to have epidemiological significance, multiple infections and HPV 16 were the predominant type. HPV 58, 52 and 18 accounted for 25% of HPV infections. HPV 52, 58 and 31 constituted 30% of CIN 2/3. Conclusion: The multiplex real‐time PCR system shows a good and reliable clinical performance. This in house PCR assay is fast and cost‐effective for HPV genotyping and the detection of HPV co‐infection in the post‐HPV vaccination era.     

Real‐time PCR detection of Enterococcus faecalis associated with amyloid arthropathy

Aims: To develop a RT‐PCR method for detection of the multilocus sequence type 82 of Enterococcus faecalis associated with amyloid arthropathy (AA) in layers. Methods and Results: Bacteria were selected from lesions including AA in layers. The primers were designed based on the phosphate ATP binding cassette transporter (pstS) and xanthine phosphoribosyltransferase (xpt) genes and first tested against three isolates with known base pairs at the specific sites. Subsequently, 12 isolates were selected from our collection by one researcher, and RT‐PCR was performed blinded. The sequence type (ST) was then confirmed by multilocus sequence analysis. Two single‐nucleotide polymorphisms in the pstS and xpt genes allowed an unambiguous identification of ST82. As an alternative to DNA extraction, a boiling method for release of DNA from cells was used. Conclusions: The real‐time PCR targeting ST82 enables rapid screening of Ent. faecalis cultured from suspect cases with results available after a few hours, much faster than multilocus sequence typing and pulse field gel electrophoresis. Significance and Impact of the Study: The new method allows a rapid screening of isolates with results available after only few hours. This RT‐PCR method could be a useful tool for molecular epidemiological studies on the spread of arthropathic and amyloidogenic Ent. faecalis within and between birds more efficiently.     

Quantitative mRNA Analysis of Eight Bovine 5‐HT Receptor Subtypes in Brain,Abomasum, and Intestine by Real‐Time RT‐PCR

Abstract

Serotoninergic pathways are involved in economically important bovine gastrointestinal (GI) motility disorders such as displaced abomasum and cecal dilatation/dislocation. The existing research tools to investigate the role of serotoninergic pathways in such disorders in ruminants comprise functional pharmacological methods, e.g., in vitro contractility studies in tissue baths, and electromyographical recordings in vivo. However, no tools for quantification of bovine serotonin receptor [5‐hydroxytryptamine receptor (5‐HTR)] expression were available so far. This study aimed to develop real‐time RT‐PCR assays for quantitative mRNA analysis of bovine 5‐HTR subtypes. Because the bovine 5‐HTR coding sequences (CDSs) were completely unknown, multiple species (human, mouse, and rat) alignment of complete CDS was used for primer design in highly homologous regions. LightCycler real‐time RT‐PCR assays (partial CDS) for the following bovine 5‐HTR subtypes were developed and validated: 5‐HTR_1A, 5‐HTR_1B, 5‐HTR_1D, 5‐HTR_1F, 5‐HTR_2A, 5‐HTR_2B, 5‐HTR_2C, and 5‐HTR₄. Intra‐ and inter‐assay coefficients of variation (CV) for the eight established assays were small, ranging from 0.49% to 2.46%. As a first physiological application, 5‐HTR mRNA expression levels were measured in brain, abomasum, and intestine of 10 healthy, lactating dairy cows. The 5‐HTR expression was quantified by normalization to the housekeeping gene glyceraldehyde‐phosphate‐dehydrogenase (GAPDH). The 5‐HTR subtype expression levels ranged from 0.001% (5‐HTR_2C in intestine) to 1% 5‐HTR/GAPDH (5‐HTR_1B and 5‐HTR₄ in intestine). There were high variations of 5‐HTR subtype mRNA expression within tissues across receptor subtypes and within receptor subtypes across tissues. In conclusion, accurate real‐time RT‐PCR assays for quantitative analysis of bovine 5‐HTR subtype gene expression were developed and validated.     

Differentiation of velogenic, mesogenic and lentogenic strains of Newcastle disease virus by multiplex RT-PCR

A multiplex RT‐PCR technique has been developed for differentiation of velogenic, mesogenic and lentogenic pathotypes of Newcastle disease virus (NDV), using a set of three oligonucleotide primers designed from NDV genomic RNA (P1, P2 and P3). The primer pair P1 and P2 generated a RT‐PCR product of 204 bp, only with RNA from velogenic and mesogenic strains, whereas the P1 and P3 generated a 364 bp product only with RNA from mesogenic and lentogenic strains. Thirty four NDV strains, including some reference strains (known pathotypes), NDV field isolates and NDV vaccine strains, as well as other avian virus strains, were tested with multiplex RT‐PCR. All reference strains tested were differentiated in agreement with their intracerebral pathogenicity index (ICPI) values or with the pathotypes known in previous reports. The nucleotide sequence analysis of RT‐PCR products for four NDV strains was fully in agreement with the RT‐PCR characterisations of these strains. The RT‐PCR results of other avian RNA viruses further confirmed the reliability and specificity of this technique. However, the RT‐PCR failed to detect some other avian NDV, which may not originate from chicken. This multiplex RT‐PCR technique is simple and easy to perform. It could be applied not only to determine the origin of NDV, but also may be used diagnostically in molecular epidemiological analysis of ND and for prediction of pathotypes of NDV isolates.     

Comparison of real-time PCR and culture isolation in colostrum-deprived pigs immunized and challenged with Haemophilus parasuis

Aims: A real‐time PCR (RT‐PCR) based on the detection of the infB gene of Haemophilus parasuis is compared with culture isolation (Frandoloso et al., (2011) Clin Vaccine Immunol 18 , 50–58.), evaluating different subunit or commercial vaccines. Methods and Results: Samples from different tissues of 24 experimentally infected and challenged colostrum‐deprived piglets were tested. The RT‐PCR gave globally a 23·3% more of positive results than culture, and all samples being positive by culture were positive by RT‐PCR also. H. parasuis could not be cultured from any of the samples of the piglets included in the three vaccinated groups resulting in a strong protection, but it could be detected by RT‐PCR in six samples in the group immunized with the commercial vaccine, in three in that vaccinated with native proteins with affinity to porcine transferrin (NPAPT) administered intramuscularly and in only two in that immunized with NPAPT intratracheally. Conclusions: The RT‐PCR was more sensitive than culture for H. parasuis detection in the organs compared. Significance and Impact of the Study: The RT‐PCR evidenced that NPAPT vaccines were those yielding the best protection results in terms of H. parasuis clearance.     

Statistical correlation between enterovirus genome copy numbers and infectious viral particles in wastewater samples

Aims: Classic virological tests are time consuming and labour‐intensive; real‐time RT‐PCR has proven to be a fast method to detect and quantify enterovirus genomes in clinical and environmental samples. This method is unable to discriminate between infective and noninfective enterovirus particles; few clinical studies have compared real‐time RT‐PCR and viral culture. We wondered if the enterovirus genome quantification could be correlated to the infectivity. Methods and Results: We used the statistical approach to verify our hypotheses to correlate data, obtained by the standard method (most probable number of cytopathic units—MPNCU) and molecular test (real‐time RT‐PCR), on wastewater treatment plant samples. Chi‐squared test was used, considering several cut‐off values (‘50’‐‘100’‐‘200’ genome copy numbers), to determine statistical significance in comparison of the two methods. Chi‐square value was not significant when cut‐off of 50 (P = 0·103) and 100 (P = 0·178) was assumed but was significant with cut‐off of 200 (P = 0·044). Conclusion: This limit, 200 genome copy, could be used as cut‐off value to indicate enterovirus survival in environmental monitoring. Significant and Impact of the Study: To introduce a fast procedure that is able to compensate for disadvantages of cell culture method for viral environmental analyses.