Sensitive Polymerase Chain Reaction Assay Research Articles

Quantitative analysis of the mRNA expression levels of BCL2 and BAX genes in human osteoarthritis and normal articular cartilage: An investigation into their differential expression.

Osteoarthritis (OA) is primarily characterized by articular cartilage degeneration and chondrocyte loss. Although the role of apoptosis in cartilage pathobiology remains to be elucidated, the apoptotic B‑cell CLL/lymphoma 2 (BCL2) gene family is considered to be involved in OA. The purpose of the present study was to quantitatively analyze the mRNA expression profiles of the BCL2‑associated X protein (BAX) and BCL2 genes in human OA and in normal cartilage. Cartilage tissue samples were obtained from 78 patients undergoing total knee arthroplasty for OA (OA group) and orthopedic interventions for causes other than OA (control group). Total RNA was isolated from the cartilage tissue specimens and reverse transcribed into cDNA. A highly sensitive and specific reverse transcription quantitative polymerase chain reaction assay was developed for quantification of the mRNA levels of BAX and BCL2, using beta‑2 microglobulin as an endogenous control for normalization purposes. Gene expression analysis was performed using the comparative Ct (2(‑ΔΔCt)) method. The mRNA expression of BAX presented an increasing trend in the OA group compared with the control group, although without statistically significace (P=0.099). By contrast, the expression ratio of BCL2/BAX was found to be significantly decreased (2.76‑fold) in the OA group compared with the normal cartilage control group (P=0.022). A notable 4.6‑fold overexpression of median mRNA levels of BAX was also observed in patients with stage III OA compared with the control (P=0.034), while the BCL2/BAX ratio was markedly (2.5‑fold) decreased (P=0.024). A marked positive correlation was observed between the mRNA levels of BAX and BCL2 in the control group (r(s)=0.728; P<0.001), which was also present in the OA group, although to a lesser degree (r(s)=0.532; P<0.001). These results further implicate apoptosis in the pathogenesis of OA, through molecular mechanisms, which include the aberrant expression of the BCL2 gene family. Further investigation may reveal novel prognostic biomarkers and potential targets for therapeutic interventions in the early stages of OA.

Molecular detection of the avian malaria parasite Plasmodium gallinaceum in Thailand

Avian malaria is one of the most common veterinary problems in Southeast Asia. The standard molecular method for detection of the avian malaria parasite involves the phenol–chloroform extraction of parasite genomic (g)DNA followed by the amplification of parasite gDNA using polymerase chain reaction (PCR). However, the phenol–chloroform extraction method is time-consuming and requires large amounts of samples and toxic organic solvents, thereby limiting its applications for parasite detection in the field. This study aimed to compare the performance of chelex-100 resin and phenol/chloroform extraction methods for the extraction of Plasmodium gallinaceum gDNA from whole avian blood that had been dried on filter papers (a common field sampling method). The specificity and sensitivity of PCR assays for P. gallinaceum cytochrome B (cytb) and cytochrome oxidase subunit I (coxI) gene fragments (544 and 588bp, respectively) were determined, and found to be more sensitive with gDNA extracted by the chelex-100 resin method than with the phenol/chloroform method. These PCR assays were also performed to detect P. gallinaceum in 29 blood samples dried on filter papers from domestic chickens in a malaria endemic area, where the reliable identification of seven field isolates of P. gallinaceum was obtained with an accuracy of 100%. The analysis of cytb and coxI gene nucleotide sequences revealed the existence of at least two genetically distinct populations of P. gallinaceum in Thailand, both of which differed from the reference strain 8A of P. gallinaceum. In conclusion, the chelex-100 resin extraction method is a simple and sensitive method for isolating gDNA from whole avian blood dried on filter paper. Genomic DNA extracted by the chelex method could subsequently be applied for the PCR-based detection of P. gallinaceum and DNA sequencing. Our PCR assays provide a reliable diagnostic tool for molecular epidemiological studies of P. gallinaceum infections in domestic chickens and wild birds.

Residual amount of HBV DNA in serum is related to relapse in chronic hepatitis B patients after cessation of nucleos(t)ide analogs.

To investigate the relationship between relapse and the levels of the residual amount of HBV DNA in serum at cessation in chronic hepatitis B patients meeting 2008 Asian Pacific Association for the Study of the Liver (APASL) nucleos(t)ide analogs (NAs) cessation criteria. A total of 72 chronic hepatitis B patients who took NAs and had reached 2008 APASL cessation criteria entered the study. Patients were followed up for 6 months or longer after antiviral therapy was stopped. Serum HBV DNA level at cessation was detected by a highly sensitive polymerase chain reaction assay with detection limitation of 2 IU/mL. Of all the 72 patients, 42 patients (65.3%) relapsed after NA cessation. The detectable rate of the trace amount of HBV DNA at cessation was 41.7% by highly sensitive polymerase chain reaction reagents. The detectable rate of patients with consolidation treatment duration of <18 months was higher than that with consolidation duration of ≥18 months (47.5% vs. 15.4%, P=0.034), and the detectable rate of patients with HBeAg seroconversion within 6 months of treatment was lower than that of ≥6 months (25.0% vs. 61.5%, P=0.036). The residual amount of HBV DNA and detectable rate at cessation showed significant differences between relapsed and nonrelapsed patients (130.4±420.90 vs 44.6±155.16 IU/mL, P=0.004; 55.3% vs. 16.0%, P=0.001). The cutoff value predicting relapse was 2.24 IU/mL, with a sensitivity of 0.553 and specificity of 0.840. Residual amount of HBV DNA in serum at NA cessation is associated with HBV relapse. The cutoff value predicting relapse was 2.24 IU/mL, with a sensitivity of 0.553 and specificity of 0.840.

PCR-Mediated Detection of Endophytic and Phytopathogenic Fungi from Needles of the Japanese Black Pine, &amp;lt;i&amp;gt;Pinus thunbergii&amp;lt;/i&amp;gt;

A specific and sensitive polymerase chain reaction (PCR) assay based on the internal transcribed spacer (ITS) region of rDNA sequences was developed to detect endophytic and phytopathogenic fungi from needles of the Japanese black pine, Pinus thunbergii. Sequences of the ITS regions of Lophodermium conigenum, Lecanosticta acicola, Pestalotiopsis neglecta, Rhizosphaera kalkhoffii, and Septorioides pini-thunbergii were compared, and each specific primer pair for these species was designed. First, the designed primer pairs were tested for their specificity to detect each species. A PCR product was amplified only each combination of species and its specific primer pair, confirming the specificity of the designed primer pairs. These primer pairs were also tested on DNA extracted from the needles of P. thunbergii. The PCR products were amplified not only in needles with lesions but also in healthy needles without symptoms. Furthermore, several endophytic and phytopathogenic fungi could be simultaneously detected from the same region in a needle. The PCR-mediated detection method developed in this study will be a valuable tool for the detection of the endophytic and phytopathogenic fungi, not only as a rapid diagnostic tool for early detection but also for monitoring variations in both the quality and quantity of the endophytic and phytopathogenic fungi in needles in Japanese black pines.

Lack of persistent hepatitis E virus infection as a cause for unexplained transaminase elevation in renal transplant recipients in India.

Hepatitis E virus (HEV) infection is highly endemic in India, being the most common cause of acute hepatitis; however, no case of chronic infection has been reported. All the human isolates of HEV from India till date have belonged to genotype 1. In contrast, in non-endemic areas, genotype 3 is the most prevalent, and persistent HEV infection has been reported among solid-organ transplant recipients. Whether persistent infection occurs with genotype 1 HEV is unclear. We therefore looked for evidence of HEV infection among renal transplant recipients with elevated alanine transaminase (ALT). Renal transplant recipients receiving immunosuppressive therapy were screened for ALT levels, irrespective of time duration since renal transplant. For those with ALT levels equal to or exceeding 50IU/mL on at least two occasions ≥3weeks apart, serum was tested for HEV RNA using a sensitive real-time reverse transcription polymerase chain reaction assay. For those testing positive, HEV genotyping and follow up for duration of viral persistence were planned. Of the 275 patients studied, 49 (17.8%, 44 male, median age = 39.5years) had elevated ALT levels (median = 62 [range = 50-477] IU/L). None of these 49 patients had detectable HEV RNA in the serum using an assay with detection sensitivity of 300 copies of RNA/mL of specimen. Our data indicate that persistent HEV infection is an infrequent cause of ALT elevation in Indian renal transplant recipients who are receiving immunosuppressive drugs. This suggests that infection with genotype 1 HEV may have either no or low potential to cause persistent infection.

Development of a Quantitative Polymerase Chain Reaction Detection Protocol for Cercospora kikuchii in Soybean Leaves and Its Use for Documenting Latent Infection as Affected by Fungicide Applications.

Cercospora leaf blight (CLB) of soybean, caused by Cercospora kikuchii, is a serious disease in the southern United States. A sensitive TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was developed to specifically detect and quantify C. kikuchii in naturally infected soybean plants. The sensitivity was 1 pg of genomic DNA, which was equivalent to about 34 copies of genome of C. kikuchii. Using this qPCR assay, we documented a very long latent infection period for C. kikuchii in soybean leaves beginning at the V3 growth stage (as early as 22 days after planting). The levels of biomass of C. kikuchii remained low until R1, and a rapid increase was detected from the R2/R3 to R4/R5 growth stages shortly before the appearance of symptoms at R6. The efficacy of various fungicide regimens under field conditions also was evaluated over a 3-year period using this qPCR method. Our results showed that multiple fungicide applications beginning at R1 until late reproductive stages suppressed the development of C. kikuchii in leaves and delayed symptom expression. Different fungicide chemistries also had differential effects on the amount of latent infection and symptom expression during late reproductive growth stages.

Occult hepatitis B virus infection in liver transplant patients in a Brazilian referral center

Estimates of occult hepatitis B virus (HBV) infection prevalence varies among different studies depending on the prevalence of HBV infection in the study population and on the sensitivity of the assay used to detect HBV DNA. We investigated the prevalence of occult HBV infection in cirrhotic patients undergoing liver transplantation in a Brazilian referral center. Frozen liver samples from 68 adults were analyzed using a nested polymerase chain reaction assay for HBV DNA. The specificity of the amplified HBV sequences was confirmed by direct sequencing of the amplicons. The patient population comprised 49 (72.1%) males and 19 (27.9%) females with a median age of 53 years (range=18-67 years). Occult HBV infection was diagnosed in three (4.4%) patients. The etiologies of the underlying chronic liver disease in these cases were alcohol abuse, HBV infection, and cryptogenic cirrhosis. Two of the patients with cryptic HBV infection also presented hepatocellular carcinoma. Markers of previous HBV infection were available in two patients with occult HBV infection and were negative in both. In conclusion, using a sensitive nested polymerase chain reaction assay to detect HBV DNA in frozen liver tissue, we found a low prevalence of occult HBV infection in cirrhotic patients undergoing liver transplant, probably due to the low prevalence of HBV infection in our population.

Detection of intrahepatic total and covalently closed circular DNA by a sensitive and specific quantitative PCR assay and its clinical significance

Background: CccDNA serves as a template for the production of HBV pregenomic-RNA and shows viral replication activity. It is responsible from the persistence of HBV during antiviral therapy. We report total-DNA and cccDNA levels in the liver biopsy samples of 6 chronic hepatitis B(CHB) patients during different phases of undergoing antiviral therapy by a sensitive and specific quantitative real-time polymerase chain reaction(PCR) assay. Methods & Materials: All the patients had undetectable serum HBV-DNA at the time of biopsy. Each patient underwent a liver biopsy, and a small piece of the biopsy specimens at least 2 mm in length was sent to labaratory in 0.9% NaCl solution. DNA was extracted from tissue using Alkaline phenol-chloroform- izoamyl alcohol process. PCR amplification was performed by TaqMan real-time PCR method. Primers and probes for intrahepatic total-DNA, cccDNA and lamivudine-resistance were designed using Oligoware 1.0 software program. ABI PRISM 7700 Sequence Detection System was used for all the analysis. Results: First patient was recieving lamivudine(LAM) for 5 years. Second and third patient recieving LAM for 38 and 42 months had elevated transaminase for the last 14 and 18 months despite undetectable serum HBV-DNA. Fourth patient was recieving LAM add-on adefovir dipivoxil for 63 months following 5 years of LAM. Fifth patient was recieving LAM add-on tenofovire disoproxil fumarat(TDV) for 3 months following 9 years of LAM. Sixth patient was recieving TDV for 3 months following one year of pegylated-IFN. Intrahepatic total\cccDNA levels in patients were 5,6x106\6,4x104, 8,8x107\4x104, 4,9x106\9,5x104, 5,5x106\2,7x104, 5,1x103\-, 9,2x107\1,1x104 coppies\mg.tissue, respectively. Although all the patients except first and second had normal levels of serum transaminase as well as undetectable serum HBV-DNA at the time of biopsy, YMDD mutations were identified in all except fourth and sixth one, then treatment regimens were reconstructed. Conclusion: Our results show that cccDNA persists in hepatocyte nucleus, even in with serological evidence of viral clearance under the long-term antiviral theraphy and its detection provides valuable information for the emergence of drug-resistance to manage treatment early. Investigating the intrahepatic forms of HBV-DNA is a good implementation for evidence-based medicine as an alternative in clinical practice. Therefore it is important to develop standardized assays to be used on clinical samples.

Editorial Commentary: Xenodiagnosis for Posttreatment Lyme Disease Syndrome: Resolving the Conundrum or Adding to It?

Editorial Commentary: Xenodiagnosis for Posttreatment Lyme Disease Syndrome: Resolving the Conundrum or Adding to It?

Burkholderia pseudomallei in soil samples from an oceanarium in Hong Kong detected using a sensitive PCR assay

Melioidosis, caused by Burkholderia pseudomallei, is an emerging infectious disease with an expanding geographical distribution. Although assessment of the environmental load of B. pseudomallei is important for risk assessment in humans or animals in endemic areas, traditional methods of bacterial culture for isolation have low sensitivities and are labor-intensive. Using a specific polymerase chain reaction (PCR) assay targeting a Tat domain protein in comparison with a bacterial culture method, we examined the prevalence of B. pseudomallei in soil samples from an oceanarium in Hong Kong where captive marine mammals and birds have contracted melioidosis. Among 1420 soil samples collected from various sites in the oceanarium over a 15-month period, B. pseudomallei was detected in nine (0.6%) soil samples using bacterial culture, whereas it was detected in 96 (6.8%) soil samples using the specific PCR assay confirmed by sequencing. The PCR-positive samples were detected during various months, with higher detection rates observed during summer months. Positive PCR detection was significantly correlated with ambient temperature (P<0.0001) and relative humidity (P=0.011) but not with daily rainfall (P=0.241) or a recent typhoon (P=0.787). PCR-positive samples were obtained from all sampling locations, with the highest detection rate in the valley. Our results suggest that B. pseudomallei is prevalent and endemic in the oceanarium. The present PCR assay is more sensitive than the bacterial culture method, and it may be used to help better assess the transmission of melioidosis and to design infection control measures for captive animals in this unique and understudied environment.

Decreased Growth Rate of P. falciparum Blood Stage Parasitemia With Age in a Holoendemic Population

In malaria holoendemic settings, decreased parasitemia and clinical disease is associated with age and cumulative exposure. The relative contribution of acquired immunity against various stages of the parasite life cycle is not well understood. In particular, it is not known whether changes in infection dynamics can be best explained by decreasing rates of infection, or by decreased growth rates of parasites in blood. Here, we analyze the dynamics of Plasmodium falciparum infection after treatment in a cohort of 197 healthy study participants of different ages. We use both polymerase chain reaction (PCR) and microscopy detection of parasitemia in order to understand parasite growth rates and infection rates over time. The more sensitive PCR assay detects parasites earlier than microscopy, and demonstrates a higher overall prevalence of infection than microscopy alone. The delay between PCR and microscopy detection is significantly longer in adults compared with children, consistent with slower parasite growth with age. We estimated the parasite multiplication rate from delay to PCR and microscopy detections of parasitemia. We find that both the delay between PCR and microscopy infection as well as the differing reinfection dynamics in different age groups are best explained by a slowing of parasite growth with age.

Polymerase Chain Reaction Test for Clostridium difficile Toxin B Gene Reveals Similar Prevalence Rates in Children With and Without Inflammatory Bowel Disease

Clinicians often evaluate for Clostridium difficile infection (CDI) in patients with inflammatory bowel disease (IBD) presenting with exacerbations. A highly sensitive polymerase chain reaction (PCR) test for the toxin B gene of C difficile is increasingly used to diagnose CDI. The aim of this study was to determine the prevalence of positive C difficile PCR results in children and young adults with and without active IBD compared with patients with non-IBD gastrointestinal disease. Fecal samples were obtained from patients with ulcerative colitis (UC, n = 76) or Crohn disease (CD, n = 69) and 51 controls followed in our gastroenterology program. Samples were analyzed for C difficile using a PCR test for the C difficile toxin B gene (BD GeneOhm Cdiff assay). Proportions of positive tests in each group were compared using the Pearson χ2 test. The prevalence of positive PCR results was 11.6% in patients with CD, 18.4% in patients with UC, and 11.8% in controls (P = 0.25). There were no significant differences in the prevalence of positive C difficile results among patients with IBD with and without active disease or among patients with and without diarrhea. Positive C difficile PCR results occur with similar frequency in patients with IBD with and without active disease and in patients with other gastrointestinal diseases. A positive result in a highly sensitive PCR assay that detects low copy numbers of a toxin gene in C difficile may reflect colonization in a subset of patients with IBD, confounding clinical decision making in managing disease exacerbations.

Vector Sequences Are Not Detected in Tumor Tissue from Research Subjects with Ornithine Transcarbamylase Deficiency Who Previously Received Adenovirus Gene Transfer

A 66-year-old woman heterozygous for a mutation in the ornithine transcarbamylase gene (Otc) participated in a phase I gene therapy trial for OTC deficiency. She received an adenovirus (Ad) vector expressing the functional OTC gene by intraportal perfusion. Fourteen years later she developed and subsequently died of hepatocellular carcinoma. A second subject, a 45-year-old woman, enrolled in the same trial presented with colon cancer 15 years later. We sought to investigate a possible association between the development of a tumor and prior adenoviral gene transfer in these two subjects. We developed and validated a sensitive nested polymerase chain reaction assay for recovering recombinant Ad sequences from host tissues. Using this method, we could not detect any Ad vector DNA in either tumor or normal tissue from the two patients. Our results are informative in ruling out the possibility that the adenoviral vector might have contributed to the development of cancer in those two subjects.

Virologic Evidences of Active Herpesviridae Infection in Children With Guillain-Barre Syndrome

Background: The etiology of the Guillain-Barre syndrome (GBS) is not clear; in many cases, GBS is triggered by a bacterial or viral illness. Objectives: To evaluate the role of active infection with Herpesviridae group and its association with this syndrome we have investigated the cerebrospinal fluid (CSF) and serum of patients with GBS for detection of these viruses. Patients and Methods: In a prospective and cross sectional study, CSF and serum samples were obtained from 15 consecutive patients admitted with GBS in the department of neurology in a university-affiliated children’s hospital in Tehran. Patients had been diagnosed with GBS based on clinical and paraclinical findings. The level of IgM and IgG antibodies to Herpes simplex virus (HSV), Epstein–Barr virus (EBV), and Cytomegalovirus (CMV) were tested by enzyme linked immunosorbent assay, (ELISA) in CSF and serum. A highly sensitive polymerase chain reaction (PCR) assay was also used for virus detection in all samples. Results: We detected viral DNA in 3 of 15 patients revealing current systemic or central nervous system (CNS) virus infection; in 2 cases CMV PCR had positive results in serum and in one case HSV PCR had positive findings in CNS. Conclusions: The clinical significance of this finding has yet to be elucidated but we showed footprint of Herpes viruses in some patients with definite GBS.

Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Based on use of a loop-mediated isothermal amplification (LAMP) identification protocol, this study attempted to detect Lactococcus garvieae in fish by using primer sets designed from an L. garvieae alpha/beta fold family hydrolase gene. Reaction time and temperatures were optimized for 60 min at 60°C with the resulting amplicons visualized by adding SYBR Green I to the reaction tube. The assay specificity was assessed using 45 different bacterial strains. Positive results were observed in all 30 L. garvieae isolates from various aquatic animals. No false-positive results were observed in 15 non-L. garvieae strains. The detection limit of the LAMP assay was 10-fold more sensitive than the conventional polymerase chain reaction (PCR) targeting 16S rDNA when using purified L. garvieae DNA. The detection limit of the LAMP assay was approximately 300 colony-forming units (CFU) using crude bacterial lysates, 100-fold more sensitive than PCR. Furthermore, L. garvieae in spleen, kidney and brain of experimentally challenged tilapia and grey mullet were detected using this optimized LAMP assay. Results of this study demonstrate the effectiveness of LAMP in providing a rapid yet simple test for detecting L. garvieae in fish.

Detection of the pandemic H1N1/2009 influenza A virus by a highly sensitive quantitative real-time reverse-transcription polymerase chain reaction assay

A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin (HA) genes of the pandemic H1N1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic H1N1/2009 virus infection.

Rapid and sensitive detection of Curvularia lunata associated with maize leaf spot based on its Clg2p gene using semi-nested PCR

Curvularia lunata (Wakker) Boed, the causative agent of Curvularia leaf spot in maize, was determined according to conidiophore and conidium morphology in a previous study. In the current study, a sensitive polymerase chain reaction assay was developed for the detection of C.lunata. Two specific forward (ClgD1/ClgD2) and one reverse primers (ClgD3) were designed based on a Ras-related (Clg2p) gene. Eight C.lunata isolates that represent different virulent strains in maize, six other Curvulariaspp., and 22 fungal plant pathogens were used to test the specificity of the primers. PCR amplification using ClgD1/ClgD3 as the first-round primers resulted in an 870-bp band from the C.lunata isolates. The detection sensitivity using ClgD1/ClgD3 was 100pg of genomic DNA. In the second round of PCR, a 1:50 dilution of the first-round PCR products was used as a template with the ClgD2/ClgD3 primer pair, which increased the detection sensitivity to 1fg. This semi-nested PCR procedure could also be used to detect C.lunata from infected maize leaves. The proposed PCR-based assay may be used for diagnosing and monitoring maize Curvularia leaf spot. The semi-nested PCR assay may provide researchers and laboratory technologists a tool to rapidly detect C. lunata, which causes maize Curvularia leaf spot, compared with histological examination.

Quantitative PCR assay to determine prevalence and intensity of MSX (Haplosporidium nelsoni) in North Carolina and Rhode Island oysters Crassostrea virginica

The continuing challenges to the management of both wild and cultured eastern oyster Crassostrea virginica populations resulting from protozoan parasites has stimulated interest in the development of molecular assays for their detection and quantification. For Haplosporidium nelsoni, the causative agent of multinucleated sphere unknown (MSX) disease, diagnostic evaluations depend extensively on traditional but laborious histological approaches and more recently on rapid and sensitive (but not quantitative) end-point polymerase chain reaction (PCR) assays. Here, we describe the development and application of a quantitative PCR (qPCR) assay for H. nelsoni using an Applied Biosystems TaqMan® assay designed with minor groove binder (MGB) probes. The assay was highly sensitive, detecting as few as 20 copies of cloned target DNA. Histologically evaluated parasite density was significantly correlated with the quantification cycle (Cq), regardless of whether quantification was categorical (r2 = 0.696, p < 0.0001) or quantitative (r2 = 0.797, p < 0.0001). Application in field studies conducted in North Carolina, USA (7 locations), revealed widespread occurrence of the parasite with moderate to high intensities noted in some locations. In Rhode Island, USA, application of the assay on oysters from 2 locations resulted in no positives.

Real-time PCR detection of Ochroconis lascauxensis involved in the formation of black stains in the Lascaux Cave, France

A real-time Polymerase Chain Reaction (PCR) assay was developed to detect and quantify Ochroconis lascauxensis in the Lascaux Cave in France. This fungus is the principal causal agent of the black stains threatening the Paleolithic paintings of this UNESCO World Heritage Site. The black stains outbreak could not be stopped in spite of using intensive biocide treatments. A sensitive and time-saving protocol is needed for determining the extent of the colonization. Sets of primers that target the ITS and RPB2 regions were designed and evaluated for specificity against O. lascauxensis. Genomic DNA extracted from five species of Ochroconis and 13 other fungal species frequently isolated from caves were used to test the specificity of each primer set. The specific and sensitive real-time PCR assay using the primers 347F/493R targeting a 147-bp fragment from the RPB2 gene was useful for quantifying the presence of O. lascauxensis in the stains on the walls, sediments and air of the cavity. The results confirmed the association of this fungus with the black stains and its wide dissemination in all cave compartments. The suitability of this method for monitoring fungal outbreaks in cave environments is discussed.

Prevalence and molecular epidemiology of human coronavirus HKU1 in patients with acute respiratory illness.

In 2005, human coronavirus HKU1 (HCoV‐HKU1) was isolated and identified from a 71‐year‐old man with pneumonia in Hong Kong. To identify and classify genotypes of HCoV‐HKU1 in Korea, a sensitive, specific, and quantitative real‐time polymerase chain reaction (PCR) assay was developed and analyzed the sequences of HCoV‐HKU1 isolated in Korea. A total of 1,985 respiratory specimens taken from patients with acute respiratory illness were tested for HCoV‐HKU1 from January 2007 to May 2008. The major clinical symptoms associated with HCoV‐HKU1 infection were examined statistically and sequence variations of the RNA‐dependent RNA polymerase (RdRp), spike, and nucleocapsid genes were also analyzed. Fifty cases (2.5%) HCoV‐HKU1 were identified by real‐time PCR and viral loads ranged from 6.7 × 104 to 1.6 × 109 copies/ml. The clinical symptoms of HCoV‐HKU1 infection included rhinorrhea (72%), cough (64%), nasal congestion (56%), fever (32%), sputum (30%), sore throat (18%), chills (16%), postnasal discharge (14%), and tonsillar hypertrophy (10%). There was a seasonal distribution of HCoV‐HKU1 infection, peaking in winter and spring. Both genotypes A and B were detected but no recombination between them was found. This is the first report on the identification and genotyping of HCoV‐HKU1 as a causative agent of acute respiratory illness in Korea. The data suggest that at least two genotypes, A and B, of HCoV‐HKU1 with scattered silent mutations were circulating in Korea from 2007 to 2008. J. Med. Virol. 85:309–314, 2013. © 2012 Wiley Periodicals, Inc.