Comparison of real-time and nested PCR assays for detection of herpes simplex virus DNA

We performed a real-time PCR assay to detect herpes simplex virus (HSV) DNA, and compared it prospectively with a nested PCR assay in 164 clinical samples (109 cerebrospinal fluid and 55 sera) from patients suspected of having neonatal HSV infection or HSV encephalitis. In 25 of 164 samples, HSV DNA was detected by the nested PCR assay. All samples positive for HSV DNA in the nested PCR assay were also positive in the real-time PCR assay, and all but two samples negative for HSV DNA in the nested assay were negative in the real-time assay. The real-time PCR assay thus had a sensitivity of 100% and a specificity of 99%, when compared with the nested assay. Sequential assays in a case of disseminated HSV showed that a decrease in HSV DNA paralleled clinical improvement. Quantification of HSV DNA by real-time PCR was useful for diagnosing and monitoring patients with HSV encephalitis and neonatal HSV infection.     

Multiplex real-time PCR assay for simultaneous detection of Omphalotus guepiniformis and Lentinula edodes

A rapid multiplex real-time PCR assay was developed to achieve highly specific, simultaneous detection of two kinds of mushrooms, Omphalotus guepiniformis and Lentinula edodes. Primers and TaqMan minor groove binder probes were designed according to the internal transcribed spacers 1-5.8S region of rDNA and evaluated by the specificity for fruiting bodies of 17 O. guepiniformis, 16 L. edodes and samples from 57 other species. DNA extracts of all the target species had positive signals with no cross-reaction, the limit of detection being 0.00025 ng of DNA. Threshold cycle (Ct) values for raw and processed fruiting bodies and for fruiting bodies (1% (w/w)) mixed with foodstuffs or artificial gastric juice contents ranged from 17.16 to 26.60 for both examined species. This new assay proved specific to the target species, highly sensitive, and applicable to processed food samples and gastric juice contents, making it useful for rapidly identifying O. guepiniformis and L. edodes.     

Multiplex real-time PCR for exhaustive detection of diarrhoeagenic Escherichia coli

Aims:  The source and routes of diarrhoeagenic Escherichia coli (DEC) have not been clarified because it is difficult to detect these organisms in samples with numerous coliform bacteria. We have developed multiplex real-time PCR assays for exhaustive detection of DEC.
Methods and Results:  Primers and TaqMan probes were designed to amplify and quantify one gene ( eae , stx1 , stx2 , elt , est , virB , aggR , astA, and afaB ) from each of seven pathotypes of DEC, in duplex or triplex reactions under the same PCR cycling conditions. Specificity was confirmed using 860 strains including 88 DEC strains. The fluorescence threshold cycle and DNA concentrations correlated with decision coefficients of more than 0·99. Subsequently, meat samples and enrichment broths were spiked with DEC and the assays used to detect the genes. The detection limits varied from 7·1 × 10² to 1·1 × 10⁴ CFU ml⁻¹, depending on the target genes. All meat samples spiked with a variety of DEC (more than 10 CFU 10 g⁻¹) were found to be positive by the method.
Conclusions:  The present system allows for the efficient and simultaneous determination of various DEC pathotypes.
Significance and Impact of the Study:  This system makes epidemiological investigations for DEC sensitive and quick, and is a useful tool to clarify the source and routes of DEC.     

Optimisation of a quantitative polymerase chain reaction-based strategy for the detection and quantification of human herpesvirus 6 DNA in patients undergoing allogeneic haematopoietic stem cell transplantation

Human herpesvirus 6 (HHV-6) may cause severe complications after haematopoietic stem cell transplantation (HSCT). Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases, constitute essential measures to improve outcomes. A prospective survey on the incidence and clinical features of HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the impact of this infection on HSCT outcome remains unclear. A rapid test based on real-time quantitative polymerase chain reaction (qPCR) has been optimised to screen and quantify clinical samples for HHV-6. The detection step was based on reaction with TaqMan^® hydrolysis probes. A set of previously described primers and probes have been tested to evaluate efficiency, sensitivity and reproducibility. The target efficiency range was 91.4% with linearity ranging from 10-10⁶ copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of plasma. The qPCR assay developed in the present study was simple, rapid and sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this test may be useful as a practical tool to help elucidate the clinical relevance of HHV-6 infection and reactivation in different scenarios and to determine the need for surveillance.     

Multiplex real-time PCR for detection of deletions and duplications in dystrophin gene

Genetic testing of Duchenne and Becker muscular dystrophies (DMD/BMD) is a difficult task due to the occurrence of deletions or duplications within dystrophin (DMD) gene that requires dose sensitive tests. We developed three multiplex quantitative real-time PCR assays for dystrophin exon 5, 45, and 51 within two major hotspots of deletion/duplication. Each exon was co-amplified with a reference X-linked gene and the copy number of the target fragment was calculated by comparative threshold cycle method (delta deltaC(t)). We compared the performance of this method with previously described end-point PCR fluorescent analysis (EPFA) by studying 24 subjects carrying DMD deletions or duplications. We showed that Q-PCR is an accurate and sensitive technique for the identification of deletions and duplications in DMD/BMD. Q-PCR is a valuable tool for independent confirmation of EPFA screening, particularly when deletions/duplications of single exons occur or for rapid identification of known mutations in at risk carriers.     

Multiplex PCR for rapid detection of minute virus of mice,bovine parvovirus,and bovine herpesvirus during the manufacture of cell culture-derived biopharmaceuticals

Validation of viral safety is essential in ensuring the safety of mammalian cell culture-derived biopharmaceuticals since numerous adventitious viruses get contaminated during the manufacturing process. In particular, Chinese hamster ovary (CHO) cells are highly susceptible to minute virus of mice (MVM), bovine parvovirus (BPV), and bovine herpesvirus (BHV). Therefore, viral detection during CHO cell culturing is necessary to ensure the safety of biopharmaceuticals against viruses. In this study, a multiplex PCR assay was developed and subsequently evaluated for its effectiveness to simultaneously detect MVM, BPV and BHV during the manufacture of cell culture-derived biopharmaceuticals. Specific primers for MVM, BPV, and BHV were selected, and a multiplex PCR was optimized. The sensitivity of the assay was 6.49 × 10¹ TCID₅₀/mL for MVM, 7.23 × 10² TCID₅₀/mL for BPV, and 5.80 × 10¹ TCID₅₀/mL for BHV. The multiplex PCR assay was very specific to MVM, BPV, and BHV and was subsequently applied to the validation of CHO cells artificially infected with each virus. It could detect each viral DNA from CHO cells as well as culture supernatants. Therefore, we concluded that the multiplex PCR assay is invaluable for detecting adventitious viruses during the manufacture of cell culture-derived biopharmaceuticals.     

Development of real-time PCR array for simultaneous detection of eight human blood-borne viral pathogens

Background

Real-time PCR array for rapid detection of multiple viral pathogens should be highly useful in cases where the sample volume and the time of testing are limited, i.e. in the eligibility testing of tissue and organ donors.

Findings

We developed a real-time PCR array capable of simultaneously detecting eight human viral pathogens: human immunodeficiency virus types 1 and 2 (HIV-1 and -2), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus-1 and -2 (HTLV-1 and -2), vaccinia virus (VACV) and West Nile virus (WNV). One hundred twenty (120) primers were designed using a combination of bioinformatics approaches, and, after experimental testing, 24 primer sets targeting eight viral pathogens were selected to set up the array with SYBR Green chemistry. The specificity and sensitivity of the virus-specific primer sets selected for the array were evaluated using analytical panels with known amounts of viruses spiked into human plasma. The array detected: 10 genome equivalents (geq)/ml of HIV-2 and HCV, 50 geq of HIV-1 (subtype B), HBV (genotype A) and WNV. It detected 100–1,000 geq/ml of plasma of HIV-1 subtypes (A – G), group N and CRF (AE and AG) isolates. Further evaluation with a panel consisting of 28 HIV-1 and HIV-2 clinical isolates revealed no cross-reactivity of HIV-1 or HIV-2 specific primers with another type of HIV. All 28 viral isolates were identified with specific primer sets targeting the most conserved genome areas. The PCR array correctly identified viral infections in a panel of 17 previously quantified clinical plasma samples positive for HIV-1, HCV or HBV at as low as several geq per PCR reaction.

Conclusions

The viral array described here demonstrated adequate performance in the testing of donors’ clinical samples. Further improvement in its sensitivity for the broad spectrum of HIV-1 subtypes is under development.     

Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab diseases and pathogens

Aims: To develop a multiplex real‐time PCR assay using TaqMan probes for the simultaneous detection and discrimination of potato powdery scab and common scab, two potato tuber diseases with similar symptoms, and the causal pathogens Spongospora subterranea and plant pathogenic Streptomyces spp. Methods and Results: Real‐time PCR primers and a probe for S. subterranea were designed based on the DNA sequence of the ribosomal RNA ITS2 region. Primers and a probe for pathogenic Streptomyces were designed based on the DNA sequence of the txtAB genes. The two sets of primer pairs and probes were used in a single real‐time PCR assay. The multiplex real‐time PCR assay was confirmed to be specific for S. subterranea and pathogenic Streptomyces. The assay detected DNA quantities of 100 fg for each of the two pathogens and linear responses and high correlation coefficients between the amount of DNA and C_t values for each pathogen were achieved. The presence of two sets of primer pairs and probes and of plant extracts did not alter the sensitivity and efficiency of multiplex PCR amplification. Using the PCR assay, we could discriminate between powdery scab and common scab tubers with similar symptoms. Common scab and powdery scab were detected in some tubers with no visible symptoms. Mixed infections of common scab and powdery scab on single tubers were also revealed. Conclusions: This multiplex real‐time PCR assay is a rapid, cost efficient, specific and sensitive tool for the simultaneous detection and discrimination of the two pathogens on infected potato tubers when visual symptoms are inconclusive or not present. Significance and Impact of the Study: Accurate and quick identification and discrimination of the cause of scab diseases on potatoes will provide critical information to potato growers and researchers for disease management. This is important because management strategies for common and powdery scab diseases are very different.     

Multiplex fluorogenic real-time PCR for detection and quantification of Escherichia coli O157:H7 in dairy wastewater wetlands

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C(T)) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C(T) and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 x 10(-5) pg of E. coli O157:H7 DNA ml(-1) equivalent to approximately 6.4 x 10(3) CFU of E. coli O157:H7 ml(-1) based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were >/=3.5 x 10(4) CFU g(-1). E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g(-1) with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.     

A role for herpesvirus entry mediator as the receptor for herpes simplex virus 1 entry into primary human trabecular meshwork cells

The human eye is an important target for infection with herpes simplex virus 1 (HSV-1). Damage to cells forming the trabeculum of the eye by HSV-1 infection could contribute to the development of glaucoma, a major blinding disease. Primary cultures of human trabecular meshwork cells were used as an in vitro model to demonstrate the ability of HSV-1 to enter into and establish a productive infection of the trabeculum. Blocking of entry by anti-herpesvirus entry mediator (HVEM) antibody implicated HVEM as the major receptor for HSV-1 infection.     

Immunoperoxidase staining for the detection of herpes simplex virus antigen in cervicovaginal smears

Destained cervicovaginal smears from eight patients with herpes simplex virus (HSV) infections were stained by means of the peroxidase-antiperoxidase (PAP) technique to demonstrate the presence of the HSV type 2 (HSV-2) antigen. Positive results were obtained in six of the eight cases, with intense staining for the HSV-2-specific antigen throughout the cytoplasm and nuclei of cells having a ground-glass nuclear appearance as well as in multinucleated giant cells. Virus isolation was successfully performed for the HSV-2-positive case that also had a histologically confirmed squamous-cell carcinoma of the cervix. The combined use of cytology and the PAP staining technique was of great value in the demonstration of cervical HSV infections.     

多重实时荧光PCR检测牛、山羊和绵羊源性成分

根据牛、山羊和绵羊线粒体细胞色素b基因序列, 设计特异性引物和以不同荧光素标记的Taqman探针。通过对PCR反应体系和反应条件的优化筛选, 建立能同时鉴别牛、山羊和绵羊源性成分的多重实时荧光PCR方法。采用本文方法与国标GB/T 20190-2006方法分别对17种不同源性动物DNA和200份不同来源样品DNA进行牛羊源性成分检测, 数据显示两者检测结果符合率达100%, 特异性相当。与国标方法相比, 本试验方法不需电泳、酶切和测序, 即可在一个PCR反应中同时鉴别检测牛、山羊和绵羊3种源性成分, 检测效率提高近3倍; 灵敏度更高, 比国标方法灵敏10倍; 适用性更广, 除了饲料, 还适用于肉品、奶品、生皮和动物油脂等动物产品的牛羊源性成分检测。     

Development of a rotor-gene real-time PCR assay for the detection and quantification of Mycoplasma genitalium

We developed and validated a real-time quantitative polymerase chain reaction (qPCR) assay to determine Mycoplasma genitalium bacterial load in endocervical swabs, based on amplification of the pdhD gene which encodes dihydrolipoamide dehydrogenase, using the Rotor-Gene platform. We first determined the qPCR assay sensitivity, limit of detection, reproducibility and specificity, and then determined the ability of the qPCR assay to quantify M. genitalium in stored endocervical specimens collected from Zimbabwean women participating in clinical research undertaken between 1999 and 2007. The qPCR assay had a detection limit of 300 genome copies/mL and demonstrated low intra- and inter-assay variability. The assay was specific for M. genitalium DNA and did not amplify the DNA from other mycoplasma and ureaplasma species. We quantified M. genitalium in 119 of 1600 endocervical swabs that tested positive for M. genitalium using the commercial Sacace M. genitalium real-time PCR, as well as 156 randomly selected swabs that were negative for M. genitalium by the same assay. The M. genitalium loads ranged between < 300 and 3,240,000 copies/mL. Overall, the qPCR assay demonstrated good range of detection, reproducibility and specificity and can be used for both qualitative and quantitative analyses of M. genitalium in endocervical specimens and potentially other genital specimens.     

Localization of the gD-binding region of the human herpes simplex virus receptor, HveA

During virus entry, herpes simplex virus (HSV) glycoprotein D (gD) binds to one of several human cellular receptors. One of these, herpesvirus entry mediator A (HveA), is a member of the tumor necrosis factor receptor (TNFR) superfamily, and its ectodomain contains four characteristic cysteine-rich pseudorepeat (CRP) elements. We previously showed that gD binds the ectodomain of HveA expressed as a truncated, soluble protein [HveA(200t)]. To localize the gD-binding domain of HveA, we expressed three additional soluble forms of HveA consisting of the first CRP [HveA(76t)], the second CRP [HveA(77-120t)], or the first and second CRPs [HveA(120t)]. Biosensor and enzyme-linked immunosorbent assay studies showed that gD bound to HveA(120t) and HveA(200t) with the same affinity. However, gD did not bind to HveA(76t) or HveA(77-120t). Furthermore, HveA(200t) and HveA(120t), but not HveA(76t) or HveA(77-120t), blocked herpes simplex virus (HSV) entry into CHO cells expressing HveA. We also generated six monoclonal antibodies (MAbs) against HveA(200t). MAbs CW1, -2, and -4 bound linear epitopes within the second CRP, while CW7 and -8 bound linear epitopes within the third or fourth CRPs. None of these MAbs blocked the binding of gD to HveA. In contrast, MAb CW3 recognized a discontinuous epitope within the first CRP of HveA, blocked the binding of gD to HveA, and exhibited a limited ability to block virus entry into cells expressing HveA, suggesting that the first domain of HveA contains at least a portion of the gD binding site. The inability of gD to bind HveA(76t) suggests that additional amino acid residues of the gD binding site may reside within the second CRP.     

Multiplex PCR assay for detection of Vibrio vulnificus biotype 2 and simultaneous discrimination of serovar E strains

In the present work we develop a multiplex PCR assay for the detection and identification of the fish pathogen Vibrio vulnificus biotype 2 with discriminating potential for zoonotic strains (serovar E). The PCR assay allowed the identification of two new biotype 2 serovar E human isolates from culture collections. Finally, the multiplex was successfully applied to both diagnosis and carrier detection in field samples.