Rapid and quantitative detection of human adenovirus DNA by real-time PCR

Rapid diagnosis of human adenovirus (HAdV) infections was achieved by PCR in the recent years. However, conventional PCR has the risk of carry-over contamination due to open handling with its products, and results are only qualitative. Therefore, a quantitative "real-time" PCR with consensus primer and probe (dual fluorescence labelled, "TaqMan") sequences for a conserved region of the hexon gene was designed and evaluated. Real-time PCR detected all 51 HAdV prototypes. Sensitivity of the assay was 相似文献   

Human papillomavirus type 16 status in cervical carcinoma cell DNA assayed by multiplex PCR

Integration of human papillomavirus (HPV) DNA into host genome occurs early in cancer development and is probably an important event in malignant transformation of cervical cancer. The HPV genome integration usually disrupts E2 gene open reading frames. It results in the lack of E2 gene suppressor of the synthesis of E6 and E7 products which, in turn, leads to the overexpression of E6 and E7 genes. The oncogenic HPV types (HPV16, -18, -45, and -58) can be present as episomes or may integrate into human chromosomes. Sixty-six cervical cancer patients positive for HPV16 were tested for the presence of E6, E2, E1, and L1 genes. Multiplex PCR was carried out in all cases. Using cluster analysis, the calculated ratios of E1/E6, E2/E6, L1/E6, E1/E2, and E2/(E1*E6) gene amplification products were divided into two or three statistically different groups. These were used for statistical analysis of the prevalence of specific gene types in histological types of cancer, different levels of clinical staging, and histologically confirmed nodal metastases. The statistical analysis proved a significant correlation in the ratios of E2/E6 and E1/E2 only. The E2/E6 and E1/E2 were higher in carcinoma in situ than in advanced squamous cancers. The E2/E6 ratios were lower in higher clinical stages. The multiplex PCR estimation of the E2/E6 ratio could be a simple method for selecting patients with a high risk of a poor outcome in a standard stage-dependent treatment procedure.     

Detection of human cytomegalovirus DNA by real-time quantitative PCR

A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA. This assay was used to demonstrate a higher CMV DNA load in plasma of bone marrow transplant patients than in that of blood donors. The CMV load was higher in CMV antigen-positive patients than in antigen-negative patients.     

Integrated human papillomavirus type 16 is frequently found in cervical cancer precursors as demonstrated by a novel quantitative real-time PCR technique

In contrast to cervical cancer, integration of human papillomavirus (HPV) DNA into the host genome has been considered a rare event in cancer precursor lesions (cervical intraepithelial neoplasia [CIN]). With our new real-time PCR method, we demonstrated that integrated HPV type 16 (HPV16) is already present in CIN lesions. The physical state of HPV16 and the viral load were simultaneously detected. A unique region of the E2 open reading frame (ORF) that is most often deleted during HPV16 integration is targeted by one set of PCR primers and a probe, and another set targets the E6 ORF. In episomal form, both targets should be equivalent, while in integrated form, the copy numbers of E2 would be less than those of E6. The method was tested with DNAs from 31 cervical lesions (non-CIN to CINIII) from 24 women prospectively followed up for 10 years. This report presents viral load and integration results from the largest series of CIN lesions described to date. Only one sample contained exclusively episomal HPV16 DNA, and this lesion regressed spontaneously. Samples from another patient, with only integrated HPV16, rapidly progressed from CINI to CINIII in 2 years. In all other patients, episomal and integrated forms of HPV16 DNA were found to coexist. Rapid progression of the CIN lesions was closely associated with a heavy load of integrated HPV16. Thus, the method described here is a very sensitive tool with which to assess the physical state of HPV, which is useful in predicting disease progression.     

Rapid detection of VHL exon deletions using real-time quantitative PCR

Various types of mutations exist that exert an effect on the normal function of a gene. Among these, exon/gene deletions often remain unnoticed in initial mutation screening. Until recently, no fast and efficient methods were available to detect this type of mutation. Molecular detection methods for gene copy number changes included Southern blot (SB) and fluorescence in situ hybridisation, both with their own intrinsic limitations. In this paper, we report the development and application of a fast, sensitive and high-resolution method for the detection of single exon or larger deletions in the VHL gene based on real-time quantitative PCR (Q-PCR). These deletions account for approximately one-fifth of all patients with the von Hippel-Lindau syndrome, a dominantly inherited highly penetrant familial cancer syndrome predisposing to specific malignancies including phaeochromocytomas and haemangioblastomas. Our VHL exon quantification strategy is based on SYBR Green I detection and normalisation using two reference genes with a normal copy number, that is, ZNF80 (3q13.31) and GPR15 (3q12.1). Choice of primer sequences and the use of two reference genes appears to be critical for accurate discrimination between 1 and 2 exon copies. In a blind Q-PCR study of 29 samples, all 14 deletions were detected, which is in perfect agreement with previously determined SB results. We propose Q-PCR as the method of choice for fast (within 3.5 h), accurate and sensitive (ng amount of input DNA) exon deletion screening in routine DNA diagnosis of VHL disease. Similar assays can be designed for deletion screening in other genetic disorders.     

Rapid and sensitive detection of ostreid herpesvirus 1 in oyster samples by real-time PCR

Herpes and herpes-like virus infections have been reported in various marine mollusc species associated with high mortality rates. Following the characterisation and genome sequencing of ostreid herpesvirus 1 (OsHV-1), specific diagnostic tools have been developed based on conventional PCR techniques or in situ hybridisation. We have now developed a real-time PCR assay for rapid, sensitive and quantitative detection of OsHV-1, and compared it with a conventional PCR technique described previously. The new assay utilised SYBR((R)) Green chemistry with specific primers C(9)/C(10) targeting the C region. The melt curve analysis of OsHV-1 DNA or DNA extracted from infected material showed only one melting temperature peak (75.75+/-0.1 degrees C). The assay had a detection limit of 4 copies/microL of viral genomic DNA and a dynamic range of 5 logs. Using infected oyster samples as template, the assay was about 100-fold more sensitive than single PCR method using C(2)/C(6) primers. The assay was applied successfully for rapid diagnosis (100 min) and quantitation of OsHV-1 in different developmental stages of Crassostrea gigas. Although it already exists a competitive PCR method to quantify OsHV-1 DNA, quantitative data that will emerge in future using the new sensitive and reliable assay will illuminate aspects of pathogenesis, in particular the viral loads in asymptomatic oysters and the kinetics of infection in specific target tissues.     

Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR

Previous studies have shown that detection of cytomegalovirus (CMV) DNA in plasma is less sensitive than the antigenemia assay for CMV surveillance in blood. In 1,983 blood samples, plasma PCR assays with three different primer sets (UL125 alone, UL126 alone, and UL55/UL123-exon 4) were compared to the pp65 antigenemia assay and blood cultures. Plasma PCR detected CMV more frequently in blood specimens than either the antigenemia assay or cultures, but of the three PCR assays, the double-primer assay (UL55/UL123-exon 4) performed best with regard to sensitivity, specificity, and predictive values compared to antigenemia: 122 of 151 antigenemia-positive samples were detected (sensitivity, 80.1%), and there were 122 samples that were PCR positive-antigenemia negative (specificity, 93%). Samples with discrepant results had a low viral load (median, 0.5 cells per slide; 1,150 copies per ml) and were often obtained from patients receiving antiviral therapy. CMV could be detected by other methods in 15 of 29 antigenemia positive-PCR negative samples compared to 121 of 122 PCR positive-antigenemia negative samples (P < 0.001). On a per-subject basis, 21 of 25 patients (antigenemia positive-PCR negative) and all 57 (PCR positive-antigenemia negative) could be confirmed at different time points during follow-up. The higher sensitivity of the double-primer assay resulted in earlier detection compared to antigenemia in a time-to-event analysis of 42 CMV-seropositive stem cell transplant recipients, and two of three patients with CMV disease who were antigenemia negative were detected by plasma PCR prior to the onset of disease. Interassay variability was low, and the dynamic range was >5 log(10). Automated DNA extraction resulted in high reproducibility, accurate CMV quantitation (R = 0.87, P < 0.001), improved sensitivity, and increased speed of sample processing. Thus, primer optimization and improved DNA extraction techniques resulted in a plasma-based PCR assay that is significantly more sensitive than pp65 antigenemia and blood cultures for detection of CMV in blood specimens.     

Rapid detection of human parechoviruses in clinical samples by real-time PCR.

BACKGROUND: Human parechoviruses (HPeVs) have been associated with severe conditions such as neonatal sepsis and meningitis in young children. Rapid identification of an infectious agent in such serious conditions in these patients is essential for adequate decision making regarding treatment and hospital stay. OBJECTIVES: We have developed an HPeV specific real-time PCR assay based on the conserved 5'untranslated region. STUDY DESIGN: To determine the detection limit of the assay, serial dilutions of HPeV in vitro RNA were tested in a background of HPeV and EV RNA-negative cerebrospinal fluid (CSF). The specificity was tested by analyzing culture isolates of HPeV 1-6, enterovirus (EV) types, human rhinoviruses (HRVs) and hepatitis A virus (HAV). To establish diagnostic relevance, 522 CSF samples from children <5 years were tested. RESULTS: The detection limit of the assay was 75 copies of HPeV cDNA per reaction. The assay was highly specific for HPeV, detecting all HPeV types. We identified HPeV infections in CSF of 20 children (3.8%), all with severe conditions such as sepsis and meningitis. CONCLUSIONS: These results suggest that HPeV screening of paediatric clinical samples should be included in viral diagnostics in suspected cases of neonatal sepsis and meningitis.     

Prognostic value of human papillomavirus types 16 and 18 DNA physical status in cervical intraepithelial neoplasia

The aim of this work was to assess the value of the physical status of human papillomavirus (HPV) DNA as a disease marker for cervical cancer development in a set of 248 DNA samples previously genotyped as HPV 16 or 18, by calculating the E2/E6 ratio through real-time PCR. There was a significant difference in integration status according to disease grade for both genotypes (p <0.001). Furthermore, especially for HPV 18, determining the DNA physical status could be a useful biomarker in predicting cervical cancer risk development, with a lower E2/E6 ratio clinically associated with the development of a precancerous lesion.     

Multiplexed, real-time PCR for quantitative detection of human adenovirus

Adenovirus infection is becoming increasingly recognized as a cause of morbidity and mortality in the immunosuppressed patient population. While early detection and quantitation of adenovirus in peripheral blood has been suggested as a means of directing and monitoring antiviral therapy in these patients, few methods have been published, particularly with respect to viral quantitation. A multiplexed real-time PCR assay was developed that can quantitatively detect a wide range of known serotypes of human adenovirus, including all of subgroups A to C. This assay was compared to a qualitative, Southern blot-based PCR assay by using 45 peripheral blood specimens from 16 patients. There was 100% concordance between the two tests in terms of qualitative results. The real-time assay detected adenovirus in patient samples at levels from <200 to 266,681 copies/ml of blood. By using control viral samples, sensitivity was demonstrated to less than 10 copies of viral genome per reaction and quantitative linearity was demonstrated from 10 to 10(6) copies of input viral DNA. Equivalent sensitivity and linearity were demonstrated for 15 different reference serotypes of adenovirus. Eleven other viral serotypes have complete target region sequence homology to one or more of the strains tested. No cross-reactivity was noted with other commonly isolated viral species. Sequence analysis showed no significant homology with any other human pathogens (bacterial or viral). This assay allows rapid, sensitive, and specific quantitation of adenovirus and may have a significant impact on the care of immunocompromised patients at risk for disseminated viral infection.     

Highly effective detection of human papillomavirus 16 and 18 DNA by a testing algorithm combining broad-spectrum and type-specific PCR

The use of a single broad-spectrum human papillomavirus (HPV) DNA-based PCR test may fail to detect lower concentrations of HPV DNA due to competition between different genotypes in mixed infections. To improve HPV detection by PCR, broad-spectrum and type-specific (TS) PCRs were combined, with a focus on HPV-16 and HPV-18. Cervical and cervicovaginal cell samples were obtained from 1,113 healthy women (age range, 15 to 25 years) participating in an HPV-16/HPV-18 candidate vaccine efficacy trial. These samples were tested by a broad-spectrum SPF₁₀ PCR-DNA enzyme immunoassay, followed by a primer SPF₁₀-based line probe assay (SPF₁₀ LiPA), and HPV-16- and HPV-18-TS PCRs. The results for the majority of the HPV-16/18 SPF₁₀ LiPA-positive samples were confirmed by TS-PCR (kappa values, 0.775 for HPV-16 and 0.785 for HPV-18). However, TS PCR revealed additional positive samples among those that contained other HPV genotypes due to competition. Conversely, SPF₁₀ LiPA identified HPV-16 or -18 in samples that remained negative by TS PCR as a result of sampling variation. Analysis of follow-up samples from more than 1,000 women confirmed that the combination of SPF₁₀-LiPA with additional HPV-16- and HPV-18-TS PCR diminishes the rate of false-negative diagnosis. The combination of broad-spectrum and TS PCRs resulted in a novel testing algorithm. This combination of assays is more accurate than either method alone, and the novel algorithm offers a highly accurate and effective method for the analysis of HPV infections.     

Rapid Staphylococcus aureus agr type determination by a novel multiplex real-time quantitative PCR assay

The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information.     

Detection and genotyping of human papillomavirus by real-time PCR assay

BackgroundDiagnosis of human papillomavirus (HPV) disease remains a challenge due to several factors related to the cost, the workload of available commercial assays to detect and genotype HPV, and to the low prevalence of infected patients.ObjectiveOur study aimed to develop a real-time PCR, based on SPF10 primers, in order to combine HPV-DNA detection and genotype identification avoiding the negative samples.Study designValidation of SYBR-green based SPF10 real-time PCR on HPV-DNA plasmids followed by the investigation of the viral status in 92 samples from oropharyngeal (94%) cutaneous biopsies (3%) and anal smears (3%) which had previously been HPV-genotyped by LiPA hybridization. In-house HPV viral loads were performed to evaluate the SPF10 real-time PCR sensitivity.ResultsData showed that 100% of HPV plasmids, assessable by LiPA hybridization, were detected and genotyped appropriately after SPF10 real-time PCR assays. These results defined a range of melting temperature peaks for HPV positivity by real-time PCR. The efficient determination of the presence of HPV-DNA by SPF10 real-time PCR was validated for 98% of clinical samples compared to commercial method. Discordant results were due to a low HPV-DNA amount and to a supplementary HPV genotype identified. The SPF10 real-time PCR sensitivity was evaluated between 1 and 10 copies/10³ cells using in-house HPV (6, 11 and 16) viral load assays.ConclusionThe real-time PCR method was efficient in combining screening and genotyping of HPV-DNA. Cost and workload reduction by SPF10 real-time PCR approach may facilitate earlier diagnosis and clinical management of HPV infected patients.     

Optimization of real-time PCR assay for rapid and sensitive detection of eubacterial 16S ribosomal DNA in platelet concentrates

A real-time PCR assay was developed for rapid detection of eubacterial 16S ribosomal DNA in platelet concentrates. The sensitivity of this assay can be hampered by contaminating DNA in the PCR reagents. Digestion of the PCR reagents with Sau3AI prior to PCR amplification was effective in eliminating this contaminating DNA without affecting the sensitivity of the assay.     

Rapid detection of subtelomeric deletion/duplication by novel real-time quantitative PCR using SYBR-green dye

Telomeric chromosome rearrangements may cause mental retardation, congenital anomalies, miscarriages, and hematological malignancies. Automated detection of subtle deletions and duplications involving telomeres is essential for high-throughput screening procedures, but impractical when conventional cytogenetic methods are used. Novel real-time PCR quantitative genotyping of subtelomeric amplicons using SYBR-green dye allows high-resolution screening of single copy number gains and losses by their relative quantification against a diploid genome. To assess the applicability of the technique in the screening and diagnosis of subtelomeric imbalances, we describe here a blinded study in which DNA from 20 negative controls and 20 patients with known unbalanced cytogenetic abnormalities involving at least one or more telomeres were analyzed using a novel human subtelomere-specific primer set, producing altogether 86 amplicons, in the SYBR-green I-based real-time quantitative PCR screening approach. Screening of the DNA samples from 20 unrelated controls for copy number polymorphism do not detect any polymorphism in the set of amplicons, but single-copy-number gains and losses were accurately detected by quantitative PCR in all patients, except the copy number alterations of the subtelomeric p-arms of the acrocentric chromosomes in two cases. Furthermore, a detailed mapping of the deletion/translocation breakpoint was demonstrated in two cases by novel real-time PCR "primer-jumping." Because of the simplicity and flexibility of the SYBR-green I-based real-time detection, the primer-set can easily be extended, either to perform further detailed molecular characterization of breakpoints or to include amplicons for the detection and/or analysis of syndromes that are associated with genomic copy number alterations, e.g., deletion/duplication-syndromes and malignant cancers.     

Rapid determination of adenoviral vector titers by quantitative real-time PCR

Replication defective adenoviruses have been used as vectors in a variety of settings including gene transfer, gene manipulation, and functionality studies. A quantitative real-time PCR-based assay is described for rapid determination of physical titers of recombinant adenovirus vectors. This method is based on amplification of a 77 bp fragment located near the left end of the adenovirus type 5 genome. Evaluation of this method demonstrated that it is simple, sensitive and reproducible, and has a dynamic range of quantitation over 5 logs. This assay is applicable to purified adenovirus as well as vectors prepared by simple cell lysis procedure, requiring only a small amount of starting material. The simplicity and short turn-around time of this assay should facilitate rapid titer determination for a large collection of adenoviral vectors.     

荧光定量聚合酶链反应检测人乳头瘤病毒6/11型

目的 了解女性阴道炎患者人乳头瘤病毒6／11型（HPV6／11）的感染情况。方法 用荧光定量聚合酶链反应（FQ－PCR）技术检测129例门诊患者阴道分泌物中HPV6／11的病毒拷贝数。结果 共检出HPV6／11阳笥者47例,阳性率为36．43％;拷贝数在10^5以上者39例,占阳性者中的82．98％;40岁以上年龄组阳性率高且病毒复制量均在10^5以上。结论 中老年阴道炎患者就诊晚,感染重。FQ－PCR检测HPV敏感、快速、准确,特别是其定量特点对临床很有意义。     

Rapid detection of blaKPC carbapenemase genes by real-time PCR

Carbapenem resistance among Enterobacteriaceae is an emerging problem worldwide. Klebsiella pneumoniae carbapenemase (bla_KPC) enzymes are among the most common β-lactamases described. In this study, we report the development and validation of a real-time PCR (q-PCR) assay for the detection of bla_KPC genes using TaqMan chemistry. The q-PCR amplification of bla_KPC DNA was linear over 7 log dilutions (r² = 0.999; slope, 3.54), and the amplification efficiency was 91.6%. The q-PCR detection limit was 1 CFU, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria. Perianal/rectal swabs (n = 187) collected in duplicate from 128 patients admitted to Sheba Medical Center surgical intensive care units were evaluated for the presence of carbapenem-resistant bacteria by culturing on MacConkey agar-plus-carbapenem disks and for bla_KPC genes by q-PCR. Carbapenem-resistant organisms, all K. pneumoniae, were isolated from 47 (25.1%) of the 187 samples collected, while bla_KPC genes were detected in 54 (28.9%) of the patient samples extracted by the NucliSENS easyMAG system. Of these, seven samples were positive for bla_KPC genes by q-PCR but negative for carbapenem resistance by culture, while all samples in which no carbapenem-resistant bacteria were detected by culture also tested negative by q-PCR. Thus, the sensitivity and specificity of the q-PCR assay after extraction by the NucliSENS easyMAG system were 100% and 95%, respectively. Similar values were obtained after DNA extraction by the Roche MagNA Pure LC instrument: 97.9% sensitivity and 96.4% specificity. Overall, the bla_KPC q-PCR assay appears to be highly sensitive and specific. The utilization of q-PCR will shorten the time to bla_KPC detection from 24 h to 4 h and will help in rapidly isolating colonized or infected patients and assigning them to cohorts.     

Specific and quantitative detection of human polyomaviruses BKV and JCV by LightCycler real-time PCR.

BACKGROUND: BK virus (BKV) and JC virus (JCV) are the only two known human polyomavirus that typically establish subclinical persistent infections. In immunocompromised hosts reactivation of the JCV infection is the cause of the central nervous system disease progressive multifocal leucoencephalopathy (PML), while BKV may cause renal nephropathy and haemorrhagic cystitis. OBJECTIVES: The goal of this study was to develop a specific quantification assay for each polyomavirus by LightCycler real-time polymerase chain reaction (PCR) based on SYBR Green I detection. STUDY DESIGN: DNA fragments of 138bp and 233bp from the "large T antigen" region of JCV and BKV, respectively, were amplified. The ability of the designed primer sets to separately quantify BKV DNA or JCV DNA and the PCR efficiency were assessed on reference DNA samples. Known amounts of cloned JCV DNA and BKV DNA from TEBU-BIO nv (Boechout, Belgium) were used to generate standard curves for the quantification assays. Species-specificity of the PCR was evaluated with cloned DNA and with DNA from patient samples. RESULTS: The assay allowed a specific quantification over a 7log dynamic range. Seventeen copies each of the viral genes were reproducibly and accurately detected. The primer sets generated specific DNA fragments for each virus confirmed by agarose gel analysis and by cycle sequencing. The similarities of the amplified gene sequences by BLAST analysis were 99% and 100% for BKV and JCV, respectively. There was no cross-reactivity within the dynamic range of the standard dilutions. CONCLUSIONS: We developed LightCycler real-time PCR assay based on SYBR Green I detection that provided rapid and specific quantification of polyomavirus load.