Universal and group-specific real-time PCR diagnosis of flavescence dorée (16Sr-V), bois noir (16Sr-XII) and apple proliferation (16Sr-X) phytoplasmas from field-collected plant hosts and insect vectors

Three real‐time PCR–based assays for the specific diagnosis of flavescence dorée (FD), bois noir (BN) and apple proliferation (AP) phytoplasmas and a universal one for the detection of phytoplasmas belonging to groups 16Sr‐V, 16Sr‐X and 16Sr‐XII have been developed. Ribosomal‐based primers CYS2Fw/Rv and TaqMan probe CYS2 were used for universal diagnosis in real‐time PCR. For group‐specific detection of FD phytoplasma, ribosomal‐based primers fAY/rEY, specific for 16Sr‐V phytoplasmas, were chosen. For diagnosis of BN and AP phytoplasmas, specific primers were designed on non‐ribosomal and nitroreductase DNA sequences, respectively. SYBR^® Green I detection coupled with melting curve analysis was used in each group‐specific protocol. Field‐collected grapevines infected with FD and BN phytoplasmas and apple trees infected with AP phytoplasma, together with Scaphoideus titanus, Hyalesthes obsoletus and Cacopsylla melanoneura adults, captured in the same vineyards and orchards, were used as templates in real‐time PCR assays. The diagnostic efficiency of each group‐specific protocol was compared with well‐established detection procedures, based on conventional nested PCR. Universal amplification was obtained in real‐time PCR from DNAs of European aster yellows (16Sr‐I), elm yellows (16Sr‐V), stolbur (16Sr‐XII) and AP phytoplasma reference isolates maintained in periwinkles. The same assay detected phytoplasma DNA in all test plants and test insect vectors infected with FD, BN and AP phytoplasmas. Our group‐specific assays detected FD, BN, and AP phytoplasmas with high efficiencies, similar to those obtained with nested PCR and did not amplify phytoplasma DNA of other taxonomic groups. Melting curve analysis was necessary for the correct identification of the specific amplicons generated in the presence of very low target concentrations. Our work shows that real‐time PCR methods can sensitively and rapidly detect phytoplasmas at the universal or group‐specific level. This should be useful in developing defence strategies and for quantitative studies of phytoplasma–plant–vector interactions.     

Detection of aster yellows phytoplasma in false flax based on PCR and RFLP

False flax (Camelina sativa L.) plants were found to be infected with a yellows-type disease caused by a phytoplasma in experimental plots at the Edmonton Research station. Alberta, Canada. Typical phytoplasmas were detected in the phloem cells in ultrathin sections from leaf midrib tissues examined by electron microscopy. These observations were supported by polymerase chain reaction (PCR) using two primer pairs, R16 F2n/R2 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. Aster yellows (AY) and potato witches'-broom (PWB) phytoplasma DNA samples served as controls and were used to study group relatedness. In a direct PCR assay, DNA amplification with universal primer pair R16F2n/R2 gave the expected PCR products of 1.2 kb. Based on a nested-PCR assay using the latter PCR products as templates, and a specific primer pair, R16(1)F1/R1, designed on the basis of AY phytoplasma rDNA sequences, a PCR product of 1.1 kb was obtained from each phytoplasma-infected false flax and AY sample, but not from PWB phytoplasma and healthy controls. DNA amplification with specific primer pair R16(1)F1/R1 and restriction fragment length polymorphism indicated the presence of AY phytoplasma in the infected false flax sample. This is the first reported characterization of AY phytoplasma in false flax.     

Aster Yellows Phytoplasma Identified in Scentless Chamomile by Microscopical Examinations and Molecular Characterization

Scentless chamomile (Matricaria perforata Mérat) plants were commonly found infected with a yellows-type disease caused by phytoplasma in several fields in Alberta, Canada. Typical phytoplasmas were detected in the phloem cells in ultrathin sections from leaf, stem, root and flower petiole tissues examined by electron microscopy. Application of 4′6-diamidino-2-phenylindole- 2HCl (DAPI) staining techniques confirmed the presence of the phytoplasma in these tissues. These observations were supported by polymerase chain reaction (PCR) assays, using two primer pairs, P1/P6 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. Aster yellows and potato witches′-broom (PWB) DNA phytoplasma samples served as positive controls and were used to study group relatedness. In a direct PCR assay, DNA amplification with universal primer pair P1/P6 gave the expected PCR products of 1.5 kb. Based on a nested-PCR assay using the latter PCR products, as templates, and a specific primer pair R16(1)F1/R1 designed on the basis of AY phytoplasma rDNA sequences, a PCR product of 1.1 kb was obtained from each phytoplasma-infected chamomile and AY samples but not from PWB phytoplasma and healthy chamomile controls. DNA amplification with specific primer pair R16(1)F1/R1 and restriction fragment length polymorphism indicated the presence of AY phytoplasma in the infected scentless chamomile sample.     

First Detection of Stolbur Phytoplasma in Grapevines (Vitis vinifera cv. Merlot) Affected with Grapevine Yellows in Bulgaria

Between 2003 and 2005, a survey was conducted throughout the grape‐growing regions of Bulgaria to identify possible infection with grapevine yellows diseases, especially Flavescence dorée (FD). The samples were checked for phytoplasmas and viruses inducing similar symptoms in the Central Laboratory for Plant Quarantine. To confirm stolbur phytoplasma infection of grapevine, a multiplex nested‐PCR assay for direct detection of FD and stolbur phytoplasmas was used. Infection of grapevine with phytoplasma was detected. The disease is very common disease in Bulgaria on tomatoes, potatoes and other crops. Monitoring is being continued. This is the first report of phytoplasma‐infected grapevine in Bulgaria.     

Molecular and microscopical detection of aster yellows phytoplasma associated with infected parsnip

Typical phytoplasma yellows symptoms were observed in parsnip (Pastinaca sativa L.) plants grown around Edmonton, Alberta, Canada. Examination of ultrathin sections of leaf midribs by electron microscopy revealed numerous phytoplasma bodies localized in the phloem cells. DNA extracted from the infected leaves was amplified with a 16S rDNA universal primer pair P1/P6 giving the expected PCR product of 1.5 kb. The phytoplasma was confirmed as a member of the aster yellows (AY) group by amplification with the specific primer pair R16(1)/F1/R1 that was designed on the basis of AY phytoplasma 16S rDNA sequences. In the nested PCR assays, the expected DNA fragment of 1.1 kb was amplified with this specific primer set. Similar restriction patterns were found for the 1.1 kb PCR products of the phytoplasma isolated from parsnip and an AY phytoplasma control after digestion with restriction endonucleases AluI, HhaI, KpnI and RsaI. This is the first reported observation of aster yellows in parsnip in Canada.     

Identification of aster yellows phytoplasma in garlic and green onion by PCR-based methods

In the summer of 1999, typical yellows-type symptoms were observed on garlic and green onion plants in a number of gardens and plots around Edmonton, Alberta, Canada. DNA was extracted from leaf tissues of evidently healthy and infected plants. DNA amplifications were conducted on these samples, using two primer pairs, R16F2n/R2 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. DNA samples of aster yellows (AY), lime witches'-broom (LWB) and potato witches'-broom (PWB) phytoplasmas served as controls and were used to determine group relatedness. In a direct polymerase chain reaction (PCR) assay, DNA amplification with universal primer pair R16F2n/R2 gave the expected amplified products of 1.2 kb. Dilution (1/40) of each of the latter products were used as template and nested with specific primer pair R16(1)F1/R1. An expected PCR product of 1.1 kb was obtained from each phytoplasma-infected garlic and green onion samples, LWB and AY phytoplasmas but not from PWB phytoplasma. An aliquot from each amplification product (1.2 kb) with universal primers was subjected to PCR-based restriction fragment length polymorphism (RFLP) to identify phytoplasma isolates, using four restriction endonucleases (AluI, KpnI, MseI and RsaI). DNA amplification with specific primer pair R16(1)F1/R1 and RFLP analysis indicated the presence of AY phytoplasma in the infected garlic and green onion samples. These results suggest that AY phytoplasma in garlic and green onion samples belong to the subgroup 16Sr1-A.     

TaqMan real-time PCR versus four conventional PCR assays for detection of apple proliferation phytoplasma

A recently developed TaqMan real-time PCR assay for detection of apple proliferation phytoplasma was evaluated in comparison to four conventional PCR-based methods with the aim to assess its potential for research and routine applications. All five protocols were tested in parallel on the same DNA isolates obtained from orchard trees. The performance of the methods was evaluated by means of sensitivity, specificity, susceptibility to inhibition, handling effort, testing time, assay expenses, and potential risk for operator and environment. Compared to the conventional PCR methods, the TaqMan real-time PCR procedure combined the highest test sensitivity with the highest test specificity and was, above all, not susceptible to PCR inhibition. Furthermore, TaqMan real-time PCR had the simplest and fastest testing process, involving a minimum of handling steps. Its disadvantage is the high cost of consumables and reagents, exceeding that of a standard PCR procedure up to four-fold. However, the higher material costs could be compensated by considerably lower personnel costs and by saving expenses for hazardous waste disposal. Due to the simple testing procedure and the output of results as numeric data the TaqMan real-time PCR assay has a high potential for automation, and seems to represent the currently most suitable method for large-scale testing procedures.     

Molecular Characterization of Aster Yellows Phytoplasma Associated with Valerian and Sowthistle Plants by PCR–RFLP Analyses

In Alberta, Canada, valerian grown for medicinal purposes and sowthistle, a common weed, showed typical aster yellows symptoms. Molecular diagnosis was made using a universal primer pair (P1 / P7) designed to amplify the entire 16S rRNA gene and the 16 / 23S intergenic spacer region in a direct polymerase chain reaction (PCR) assay. This primer pair amplified the DNA samples from valerian and sowthistle and reference controls (AY‐27, CP, PWB, AY of canola, LWB). They produced the expected PCR products of 1.8 kb, which were diluted and used as templates in a nested PCR. Two primer pairs R16F2n / R2 and P3 / P7 amplified the DNA templates giving PCR products of 1.2 and 0.32 kb, respectively. No PCR product was obtained with either set of primers and DNA isolated from healthy plants. Restriction fragment length polymorphism (RFLP) was used to analyse the partial 16S rDNA sequences (1.2 kb) of all phytoplasma DNA samples after restriction with four endonucleases (AluI, HhaI, MseI and RsaI). The restriction patterns of these strains were found to be identical with the RFLP pattern of the AY phytoplasma reference control (AY‐27 strain). Based on the RFLP data, the two strains are members of subgroup A of the AY 16Sr1 group. We report here the first molecular study on the association of AY phytoplasmas with valerian and sowthistle plants.     

Molecular study of two distinct phytoplasma species associated with streak yellows of date palm in Iran

A disease with symptoms similar to palm lethal yellowing was noticed in the early 2013 in Khuzestan Province (Iran) in date palm (Phoenix dactylifera). Infected trees displaying symptoms of streak yellows and varied in the incidence and severity of yellowing. A study was initiated to determine whether phytoplasma was the causal agent. Polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) methods using universal phytoplasma primers pairs R16mF1/mR1 and M1/M2 were employed to detect putative phytoplasma(s) associated with date palm trees. Nested PCR using universal primers revealed that 40 out of 53 trees were positive for phytoplasma while asymptomatic date palms from another location (controls) tested negative. RFLP analyses and DNA sequencing of 16S rDNA indicated that the presence of two different phytoplasmas most closely related to clover proliferation (CP) phytoplasma (group 16SrVI) and ash yellows (AY) phytoplasma (group 16SrVII). Sequence analysis confirmed that palm streak yellows phytoplasmas in each group were uniform and to be phylogenetically closest to “CandidatusP. fraxini” (MF374755) and “Ca. P. trifolii” isolate Rus‐CP361Fc1 (KX773529). Result of RFLP analysis of secA gene of positive samples using TruI and TaqI endonuclease is in agreement with rDNA analysis. On this basis, both strains were classified as members of subgroups 16SrVI‐A and 16SrVII‐A. This is the first report of a phytoplasma related to CP and AY phytoplasma causing date palm yellows disease symptoms.     

Detection of lethal yellowing phytoplasma in coconut plantlets obtained through in vitro germination of zygotic embryos from the seeds of infected palms

Lethal yellowing (LY) is a disease caused by 16SrIV phytoplasmas that has devastated coconut plantations in the Americas. An alternative means of phytoplasma spread is through seeds. Therefore, we used a novel approach based on plumules from the embryos of LY‐diseased coconut palms. We cultured the plumules in vitro to determine the presence of phytoplasma DNA in the plantlets. In the first assay, 185 embryos were obtained. The results showed positive detection in 20 samples (11%) with the nested PCR and in 59 samples (32%) with the TaqMan real‐time PCR. A second assay was designed to trace plumules to their respective embryos and haustorial tissues to determine whether they had derived from an embryo with positive LY detection; a total of 124 embryos were obtained. The results showed no positive detection with the nested PCR and positive detection in 42 of the haustorial tissue samples (32%) with the TaqMan real‐time PCR. The 124 plumules isolated from the embryos were cultivated under in vitro conditions and divided into two groups. Group A was followed for shoot formation and Group B was followed to the plantlet stage. After 3 months of cultivation, 33 cultures (50%) within Group A became necrotic; the rest were analysed to evaluate LY phytoplasma DNA with the TaqMan real‐time PCR assay and 14 (42%) tested positive. After 18 months of cultivation, 20 cultures (34%) within Group B became necrotic. The rest were analysed for the detection of the LY phytoplasma DNA, and 15 and 11 (39% and 29%) of the samples tested positive with the TaqMan real‐time PCR and nested PCR assays, respectively. Blast analysis of the sequenced products revealed that the sequences showed 99% homology with LY‐phytoplasma subgroup 16SrIV‐A. The results presented here demonstrate, for the first time, the occurrence of the transmission of LY phytoplasmas from coconut embryos to plantlets.     

Heteroduplex mobility assay detects DNA mutations for differentiation of closely related phytoplasma strains

We present the first use of DNA heteroduplex mobility assay (HMA) to detect the point mutations including substitutions and deletions/insertions in 16S rDNA of aster yellows phytoplasma (AY27) and to differentiate phytoplasmas collected from field samples of clover proliferation (CP) and alfalfa witches'-broom (AWB). The phytoplasmal 16S rDNA fragment was amplified from AY27 by polymerase chain reaction (PCR) and cloned into a plasmid vector. The cloned DNA fragment was subjected to in vitro mutation to produce 1- to 4-base substitutions and 1- to 3-base deletions. The mutated 16S rDNA fragments were analyzed by HMA. The results showed that a single two-base substitution or a single-base deletion/insertion in the 529 bp DNA fragment was directly detected and that a DNA divergence at a level of as low as 0.2% was detectable by HMA. Heteroduplex mobilities were affected by the number and composition of the phytoplasma DNA bases in mismatches or gaps and were proportional to the degree of DNA divergences. Gaps caused greater retardation in heteroduplex mobility than mismatches did. HMA was highly sensitive in detecting the mixed infections of phytoplasmas. In analyses of CP and AWB field samples collected in Alberta, two CP and one AWB phytoplasma isolates were differentiated from others by HMA but not by restriction fragment length polymorphism (RFLP). Therefore, HMA provides a simple, rapid, highly sensitive and analytical method to detect and estimate the genetic divergence of phytoplasmas when other methods such as RFLP are not readily applicable.     

Amplification of plasmid DNA to detect plant pathogenic mycoplasmalike organisms

The polymerase chain reaction (PCR) was employed to develop a specific assay for plant pathogenic mycoplasmalike organisms (MLOs). A cloned fragment of a plasmid from a severe strain of western aster yellows (AY)-MLO was sequenced to identify oligonucleotide primers for PCR. Amplified DNA fragments of the predicted size were obtained from DNA extracted from plants and insects infected with pear decline MLO, beet leafhopper-transmitted virescence agent, elm yellows MLO and several AY-MLO strains. No amplification occurred from healthy leafhopper or plant DNA. The PCR-based assay was over 500 times more sensitive than a _tilized_tion-based assay which _tilized a cloned AY plasmid fragment as a probe. With the PCR-based assay, MLOs could be detected using DNA samples of leafhoppers that were only crushed and boiled in buffer. Amplification of the target DNA was confirmed by digestion of the PCR product with Mbo I which yielded predicted sized fragments for all MLO strains except Bradford AY and eastern AY. Sequencing the PCR product from elm yellows and eastern AY-MLOs revealed greater than 90% homology, and the failure to restrict the PCR product with Mbo I was due to a single base change in the restriction endonuclease site. The ability of the assay to detect a wide range of MLOs with minimal sample preparation and high sensitivity will be useful in epidemiological studies of MLO-caused diseases.     

从粪便样品中检测动物冠状病毒的分子技术研究

目的 感染冠状病毒的动物向环境排毒主要是通过粪便,建立直接从粪便样品对动物冠状病毒进行检测的分子技术具有重要的公共卫生学意义。方法 通过计算机模拟和实验方法对已报道的2对针对冠状病毒pol基因的通用引物的通用性进行了验证。不经传统的病毒分离．直接从环境样品中提取病毒RNA,通过一步法RT-PCR进行检测,并通过分子杂交和Nested PCR扩增结合TaqMan探针实时荧光检测的PCR技术．提高对冠状病毒检测的灵敏度和准确度,并对猪、禽冠状病毒感染的临床样品进行分析检测。结果 2对引物可以覆盖所有已知的冠状病毒,包括SARS,RT-PCR产物通过测序可以确定冠状病毒种类;实时荧光定量NestedPCR有很高的灵敏度,可以灵敏地检出所有供试的阳性样品,而荧光增量实时监测可以排除凝胶电泳检查的假阳性。结论 该研究为从环境中普查和鉴定冠状病毒提供了可靠的技术方法。     

Occurrence of European Stone Fruit Yellows Phytoplasma (ESFYP) Infection in Peach Orchards in Northern-Central Italy

During field surveys in 1999 and 2000 of peach orchards in Northern-Central Italy, plants of different cultivars were observed with symptoms of early leaf reddening, abnormal thickening of midribs and primary veins, autumnal growth of latent buds which produce tiny chlorotic leaves and sometimes flowers, and early phylloptosis; such symptoms, rarely seen previously on peach, are associated with European stone fruit yellows phytoplasma (ESFYP). In the orchards inspected, 1–4% of trees were affected and most were grafted on cv. G.F. 677. In most of the symptomatic samples (130 of 157 tested), only ESFYP was detected using different diagnostic methods [4',6'-diamidino-2-phenilindole, 2HCl (DAPI), polymerase chain reaction (PCR) with ribosomal and non-ribosomal primer pairs, PCR-enzyme-linked immunosorbent assay, nested-PCR]. The immunoenzymatic detection of PCR products with a pathogen-specific probe ensured fast, sensitive and specific detection of ESFYP. This is the first survey to assess the occurrence of phytoplasmas in peach orchards in Northern-Central Italy.     

Relative quantification of chrysanthemum yellows (16Sr I) phytoplasma in its plant and insect host using real-time polymerase chain reaction

A real-time polymerase chain reaction (PCR) method for the quantification of chrysanthemum yellows (CY) phytoplasma DNA in its plant (Chrysanthemum carinatum) and insect (Macrosteles quadripunctulatus) host is described. The quantity of CY DNA was measured in each run relative to the amount of host DNA in the sample. Primers and a TaqMan probe for the specific PCR amplification of phytoplasma DNA were designed on a cloned CY-specific ribosomal fragment. Primers and TaqMan probes were also designed on sequences of the internal transcribed spacer region of the insect’s ITS1 rDNA and of the plant’s 18S rDNA for amplification from C. carinatum and M. quadripunculatus, respectively. Absolute quantification of CY DNA was achieved by comparison with a dilution series of the plasmid containing a CY 16S rDNA target sequence. Absolute quantification of plant and insect DNAs was achieved by comparison with a dilution series of the corresponding DNAs. Quantification of CY DNA in relation to host DNA was finally expressed as genome units (GU) of phytoplasma DNA per nanogram of host (plant or insect) DNA. Relative quantification avoided influences due to different yields during the DNA extraction procedure. The quantity of CY DNA was about 10,000–20,000 GU/ng of plant DNA and about 30,000–50,000 GU/ng of insect DNA. The method described could be used to phytoplasma multiplication and movement in different plant and insect hosts.     

A real-time PCR for the detection of Salmonella Enteritidis in poultry meat and consumption eggs

A robust duplex 5' nuclease (TaqMan) real-time PCR was developed and in-house validated for the specific detection of Salmonella enterica subspecies enterica serovar Enteritidis in whole chicken carcass rinses and consumption eggs. The assay uses specifically designed primers and a TaqMan probe to target the Prot6e gene located on the S. Enteritidis specific 60-kb virulence plasmid. As an internal amplification control to monitor Salmonella DNA in the sample, a second primer/TaqMan probe set detects simultaneously the Salmonella specific invA gene. The assay identified correctly 95% of the 79 Salmonella Enteritidis strains tested comprising 19 different phage types. None of the 119 non-Enteritidis strains comprising 54 serovars was positive for the Prot6e gene. The assay detection probability was for 10(2) or more genome equivalents 100% and for 10 equivalents 83%. A pre-PCR sample preparation protocol including a pre-enrichment step in buffered peptone water, followed by DNA extraction was applied on low levels of artificially contaminated whole chicken carcass rinses and eggs from hens as well as 25 potentially naturally contaminated chickens. The detection limit was less than three CFU per 50 ml carcass rinse or 10 ml egg. The sensitivity and specificity compared to the traditional culture-based detection method and serotyping were both 100%. Twenty-five potentially naturally contaminated chickens were compared by the real-time PCR and the traditional cultural isolation method resulting in four Salmonella positive samples of which two were positive for the Prot6e gene and serotyped as S. Enteritidis. We show also that Salmonella isolates which have a rough lipopolysaccharide structure could be assigned to the serovar Enteritidis by the real-time PCR. This methodology can contribute to meet the need of fast identification and detection methods for use in monitoring and control measures programmes.     

A real-time PCR for SARS-coronavirus incorporating target gene pre-amplification

An enhanced polymerase chain reaction (PCR) assay to detect the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) was developed in which a target gene pre-amplification step preceded TaqMan real-time fluorescent PCR. Clinical samples were collected from 120 patients diagnosed as suspected or probable SARS cases and analyzed by conventional PCR followed by agarose gel electrophoresis, conventional TaqMan real-time PCR, and our enhanced TaqMan real-time PCR assays. An amplicon of the size expected from SARS-CoV was obtained from 28/120 samples using the enhanced real-time PCR method. Conventional PCR and real-time PCR alone identified fewer SARS-CoV positive cases. Results were confirmed by viral culture in 3/28 cases. The limit of detection of the enhanced real-time PCR method was 10(2)-fold higher than the standard real-time PCR assay and 10(7)-fold higher than conventional PCR methods. The increased sensitivity of the assay may help control the spread of the disease during future SARS outbreaks.     

Application of real-time PCR for quantitative detection of Clostridium botulinum type A toxin gene in food

The TaqMan real-time PCR method for the quantitative detection of C. botulinum type A was developed based on sequence-specific hybridization probes. The validity of this assay was verified by using 10 genera of 20 strains, including reference strains of C. botulinum types A, B, C, D, E and F. The detection limit of this assay was evaluated on C. botulinum type A, using a 10-fold dilution series of DNA and spores . The DNA and spores were detected up to level of 0.1 ng/ml and 10(2)spores/ml, respectively. Spore spiked food sample preparation prior to the real-time PCR was performed by two methods, heat treatment and GuSCN. The detection limits after heat treatment showed 10(2) spores/ml for spiked sausage slurry, and 10(3) spores/ml for spiked canned corn slurry, while detection limits after GuSCN precipitation showed 10(2) spores/ml in both sausage and canned corn. Therefore the real-time PCR assay after GuSCN precipitation is useful for the quantification of C. botulinum type A because it showed identical CT values in both pure spore solutions and food slurries. We suggest that quantitative analysis of C. botulinum type A by TaqMan real-time PCR can be a rapid and accurate assessment method for botulinal risk in food samples.     

Development of a real-time PCR assay for detection of monodon baculovirus (MBV) in penaeid shrimp

A real-time PCR method was developed to detect monodon baculovirus (MBV) in penaeid shrimp. A pair of MBV primers to amplify a 135 bp DNA fragment and a TaqMan probe were developed. The primers and TaqMan probe were specific for MBV and did not cross react with Hepatopancreatic parvovirus (HPV), White spot syndrome virus (WSSV), Infectious hypodermal and haematopoietic virus (IHHNV) and specific pathogen free (SPF) shrimp DNA. A plasmid (pMBV) containing the target MBV sequence was constructed and used for determination of the sensitivity of the real-time PCR. This real-time PCR assay had a detection limit of one plasmid MBV DNA copy. Most significantly, this real-time PCR method can detect MBV positive samples from different geographic locations in the University of Arizona collection, including Thailand and Indonesia collected over a 13-year period.     

Modification and optimization of PCR methods for detection of MIE region from human herpesvirus 5

Human herpesvirus 5 (HHV-5, formerly known as CMV) is a beta-herpesvirus widely spread within a population. Thus, HHV-5 infections are a serious matter of concern in a group of immunocompromised patients. The goal of the study was modification and optimization of conventional PCR method developed for the detection of HHV-5 DNA to the real-time variant (RTmPCR) and determination of analytical resolution of the modified methods. Thirty plasma samples were tested for the presence of HHV-5 DNA using the LightCycler system with two different methods--one with SYBR Green I fluorochrome method and second one using TaqMan fluorescent probes and a qualitative in-house gel-stained PCR assay using primers that amplify part of HHV-5 MIE gene. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of HHV-5 DNA in range between 10(0) and 10(-6). For comparison typical end-point detected PCR for cytomegalovirus detection with the same DNA dilutions was made. The sensitivity of novel method was about 100-fold higher than older one. Both LightCycler assays detected HHV-5 DNA in 27 samples, also which were negative by the gel-stained PCR. Analysis of the available clinical and serological data associated with these samples suggested that the real-time results in all of these cases were true positive. The conclusion is that real-time PCR methods are more sensitive than the conventional PCR used in this study. The additional sensitivity was valuable for detection of patients with low-copy viremia. The high level of sensitivity, specificity, accuracy, and rapidity provided by the LightCycler instrument are favorable for the use of this system in the detection of HHV-5 DNA in clinical specimens.