Quantitative Polymerase Chain Reaction (qPCR) Protocol and Applications

Leishmania braziliensis is the most prevalent agent causing cutaneous leishmaniasis (CL) in Brazil. While inflammation is a hallmark of CL, few parasites are found at the lesion site, leading to challenges regarding diagnosis. Using L braziliensis kDNA and human 18S rRNA as targets, the present study developed a quantitative polymerase chain reaction assay to determine parasite load in biopsies from patients with CL who were residing in an endemic area in northeastern Brazil. In addition, we investigated whether parasite load correlated with clinical outcome, and we observed that patients with higher parasite load were more likely to experience therapy failure. Moreover, patients with CL in the early phase of infection presented higher levels of parasite transcripts than individuals in later phases. Thus, our results suggest that parasite load as determined by quantitative polymerase chain reaction may constitute a valuable prognostic tool to aid in the determination of disease severity and treatment outcome.

ObjectivesLower limb muscle power is thought to influence outcome following total knee replacement (TKR). Post-operative deficits in muscle strength are commonly reported, although not explained. We hypothesised that post-operative recovery of lower limb muscle power would be influenced by the number of satellite cells in the quadriceps muscle at time of surgery. MethodsBiopsies were obtained from 29 patients undergoing TKR. Power output was assessed pre-operatively and at six and 26 weeks post-operatively with a Leg Extensor Power Rig and data were scaled for body weight. Satellite cell content was assessed in two separate analyses, the first cohort (n = 18) using immunohistochemistry and the second (n = 11) by a new quantitative polymerase chain reaction (q-PCR) protocol for Pax-7 (generic satellite cell marker) and Neural Cell Adhesion Molecule (NCAM; marker of activated cells).ResultsA significant improvement in power output was observed post-operatively with a mean improvement of 19.7 W (95% confidence interval (CI) 14.43 to 30.07; p < 0.001) in the first cohort and 27.5 W (95% CI 13.2 to 41.9; p = 0.002) in the second. A strong correlation was noted between satellite cell number (immunohistochemistry) and improvement in patient power output (r = 0.64, p = 0.008). Strong correlation was also observed between the expression of Pax-7 and power output (r = 0.79, p = 0.004), and the expression of NCAM and power output (r = 0.84, p = 0.001). The generic marker explained 58% of the variation in power output, and the marker of activated cells 67%.ConclusionsMuscle satellite cell content may determine improvement in lower limb power generation (and thus function) following TKR.

Rusty root rot is the most destructive soilborne disease of ginseng caused by pathogenic Ilyonectria spp., predominantly Ilyonectria robusta, in China. However, there remains no effective strategy to control the disease. Current control of the disease requires that soil and ginseng seeds and seedlings infected with I. robusta are avoided during planting. Therefore, rapid and accurate detection of I. robusta would be indispensable in disease control programs. A one-step polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) assay was developed to detect I. robusta in ginseng seeds, roots, and soil. The species-specific primers HIS H3-F and HIS H3-R, designed based on a partial histone gene sequence of I. robusta, yielded a 268-bp product using the optimized PCR and qPCR protocol. DNA of I. robusta was detected by qPCR in all diseased soil and ginseng roots and seeds resulting from artificial inoculation and sampled from natural fields. I. robusta was detected at an abundance of 1.42 fg/μl at 12 h postinoculation and 191.31 fg/μl at 7 days postinoculation in ginseng roots that showed disease symptoms. In naturally infected soil sampled from ginseng fields, pathogen abundances ranging from 13.23 to 503.39 fg/μl were detected, which were 2.04 to 11.01 times higher than those in ginseng roots. The pathogen was first detected and was more abundant on the surface of the ginseng seed coat compared with that in the seed kernel. This study provides a high-efficiency detection technique for early diagnosis of I. robusta and real-time disease prevention and control strategies.

A Multiplex Polymerase Chain Reaction Assay for Direct Detection and Differentiation of β-Hemolytic Streptococci in Clinical Samples from Horses

Locked Nucleic Acids Can Enhance the Analytical Performance of Quantitative Methylation-Specific Polymerase Chain Reaction

24 - Longer PCR Amplifications

Sensitive techniques for the detection of Plasmodium (Aconoidasida: Plasmodiidae) sporozoites in field-collected malaria vectors are essential for the correct assessment of risk for malaria transmission. A real-time polymerase chain reaction (RT-PCR) protocol targeting Plasmodium mtDNA proved to be much more sensitive in detecting sporozoites in mosquitoes than the widely used enzyme-linked immunosorbent assay targeting Plasmodium circumsporozoite protein (CSP-ELISA). However, because of the relatively high costs associated with equipment and reagents, RT-PCRs are mostly used to assess the outcomes of experimental infections in the frame of research experiments, rather than in routine monitoring of mosquito infection in the field. The present authors developed a novel mtDNA-based nested PCR protocol, modified from a loop-mediated isothermal amplification (LAMP) assay for Plasmodium recognition in human blood samples, and compared its performance with that of routinely used CSP-ELISAs in field-collected Anopheles coluzzii (Diptera: Culicidae) samples. The nested PCR showed 1.4-fold higher sensitivity than the CSP-ELISA. However, nested PCR results obtained in two laboratories and in different replicates within the same laboratory were not 100% consistent, probably because the copy number of amplifiable Plasmodium mtDNA was close in some specimens to the threshold of nested PCR sensitivity. This implies that Plasmodium-positive specimens should be confirmed by a second nested PCR to avoid false positives. Overall, the results emphasize the need to use molecular approaches to obtain accurate estimates of the actual level of Plasmodium circulation within malaria vector populations.

Hepatitis B viral infection is among the most common causes of cirrhosis and hepatocellular carcinoma and a frequent viral indication for liver transplant. Cytokine-mediated immunity plays a critical role in introducing and promoting hepatitis B virus outcomes and in graft microenvironment. Interleukin 27 is a heterodimeric cytokine and a member of interleukin-6/interleukin-12 family. Interleukin-27 shows a broad range of pro- and antiinflammatory properties and plays a determining role during immune responses in combating hepatitis B virus. Therefore, in this study, the possible association between expressions of interleukin-27 gene with hepatitis B virus infection was evaluated in liver transplant patients. In a cross-sectional study from liver transplant patients with the risk of hepatitis B virus infection who admitted to Namazi Hospital affiliated to Shiraz University of Medical Sciences, 50 patients were selected and subgrouped to 25 hepatitis B virus-infected and 25 noninfected ones between years 2011 and 2013. The 25 healthy controls also were enrolled in this study. The presence of hepatitis B virus infection was assessed using polymerase chain reaction and enzyme-linked immunosorbent assay protocols in liver transplant patients. In addition, the interleukin-27 gene expression level was analyzed using an in-house-SYBER Green real time polymerase chain reaction method. The rate of interleukin-27 gene expression level was statistically analyzed in studied patient groups and controls using the Livak (2-▵▵CT) method. The expression level of interleukin-27 gene was increased 10.27- and 2.36-fold in hepatitis B virus-infected and uninfected liver transplanted patients compared with healthy controls. Hepatitis B virus infection can lead to overexpression of interleukin-27 gene in liver transplant patients compared with uninfected ones and controls. However, further studies are needed to characterize the effective antihepatitis B virus effects of interleukin-27 in liver transplant patients.

Searching for the Answer to Irritable Bowel Syndrome in the Colonic Mucosa: SERTainty and UnSERTainty

Real-time polymerase chain reaction detection of Cryptococcus neoformans and Cryptococcus gattii in human samples

Taking advantage of the high specificity of double-dye hydrolysis-locked nucleic acid (LNA) probes (Ugozzoli et al, 2004), we developed a qualitative three-colour real-time polymerase chain reaction (PCR) assay for the genotyping of sickle cell disease (SCD). The term SCD encompasses a group of disorders associated with mutations in the Haemoglobin Beta gene (HBB). The most common forms are defined by the presence, in codon 6, of an E6V mutation (GAG > GTG; Haemoglobin S; Hb S) on both alleles (sickle cell anaemia) and co-inheritance of Hb S and Hb C (E6K mutation; GAG > AAG; Hb SC disease). The highest prevalence of SCD is found in sub-Saharan Africa (sickle-cell gene carrier prevalence varying between 5% and 40% of the population), and most affected children die in the early years of life without being diagnosed or because of non-adapted treatments (Weatherall & Clegg, 2001). The methodological approach presented here combined the use of a primer pair designed to amplify the region of interest in the HBB gene and three double-dye probes containing LNA nucleotides. Each probe was labelled with a different fluorochrome to enable the different alleles to be discriminated (Fig 1). Primers were designed with the freeware meltcalc, ver. 2.0 (http://www.meltcalc.de). The Tm of each double-dye LNA probe was calculated by using the Exiqon Tm prediction tool (http://lna-tm.com). Specificity of the probes used in this approach. 20 ng of genomic DNA from three patients homozygous for the wild-type (WT/WT), S (S/S) and C (C/C) alleles respectively was tested in this experiment. The results given by the each probe are illustrated on different panels. The Cy5 channel was applied for the analysis of the WT probe, while FAM and Yakima Yellow were used for the S and C probes, respectively. The three probes showed absolute specificity because there was no signal detected when amplified genomic DNA did not contain the nucleotide targeted by the probe. The no template control did not give any signal for the three probes either. Samples from patients that were homozygous for each of the three aforementioned alleles were used to demonstrate the specificity of the probes (Fig 1). We used unique PCR conditions on the ABI 7500 Fast machine (Applied Biosystems, Foster City, CA, USA), which generated specific results for each probe in <40 min (see Table SI, Fig S1). A cohort of 37 genomic DNA representing the different possible genotypes was used to compare our new technique with either a PCR followed by restriction endonuclease cleavage to detect the A and S alleles (Saiki et al, 1988), or an allele-specific PCR designed to detect A and C alleles (Fischel-Ghodsian et al, 1990). A 100% concordance was observed between our technology and the two published methods. Moreover, the deduced genotypes matched perfectly with the available phenotypic analysis (Gulbis et al, 2006). To investigate the sensitivity of this analysis, we tested different amounts of genomic DNA per PCR reaction. The results were perfectly interpretable from 100 to 1 ng of starting material (data not shown), and enabled the testing of prenatal diagnosis samples even when there was scarce fetal tissue (demonstrated on 18 gDNA obtained from fetal samples; data not shown). Finally, to demonstrate the robustness of our approach, we tested 10 representative samples on currently available instruments that are widely used for multicolour real-time PCR analysis. The results obtained on the LC480 (Roche, Basel, Switzerland), the iCycler (Bio-Rad, Hercules, CA, USA) and the M × 3000 p (Stratagene, La Jolla, CA, USA) were perfectly comparable from one machine to another (see Fig S1). Although a fast PCR protocol was not applicable on all these machines, a longer PCR protocol did not affect the results. In summary, we have developed a very robust and rapid method that takes advantage of the high specificity of double-dye LNA probes, which enabled the identification of the two main mutations in SCD in <40 min. The protocol developed in our laboratory is easily transferable to other real-time PCR machines. The high sensitivity of our approach represents a great advantage for the analysis of samples containing small amounts of DNA. Moreover, this single closed tube method offers another obvious advantage for prenatal diagnosis as it prevents risk of cross contamination. We gratefully acknowledge the skillful technical assistance of Sandrine Delbauve. Table SI. Oligonucleotides used for the three-colour real-time polymerase chain reaction (PCR) assay in this study. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Trichomonas vaginalis is the cause of one of the most common types of vaginitis, trichomoniasis. Clinical manifestations of symptomatic women are generally nonspecific, but include vaginal discharge, vaginitis and irritation. T. vaginalis infection has also been linked to the increased risk of human immunodeficiency virus transmission. The aim of the study was to compare a real-time polymerase chain reaction (PCR) assay with culture and wet-mount examination for the detection of T. vaginalis. This is a descriptive analytical study. Vaginal swabs from 504 female patients with suspected vaginitis were included in the study. They were subjected to culture, wet-mount microscopic examination (WM), and real-time PCR. Real-time fluorescence resonance energy transfer hybridization probe PCR assays were used in this study to test for T. vaginalis DNA, targeting the b-tubulin genes. The result of the PCR was compared with culture and wet-mount microscopy. WM were positive for T. vaginalis in 60/504 cases (11.9%) and cultures were positive for T. vaginalis in 116/504 cases (23%). Real-time PCR was done on 50/504 specimens which was randomly chosen, 33/50 (66%) cases were positive for T. vaginalis (78% sensitivity), 25/50 (50%) of them were culture and wet-mount examination positive. Of them, 1/33 (3%) were culture positive and wet examination negative, 7/33 (21%) real-time positive were negative by culture while 17/50 (34%) real-time PCR negative cases were also negative by both techniques. The time taken for PCR assay was 4 to 8 h whereas positive protozoal cultures took up to 4 days. The Real-time PCR done in this study, provide a rapid, sensitive and specific diagnosis of T. vaginalis infection.

Histoplasmosis is considered one of the most important endemic and systemic mycoses worldwide. Until now few molecular techniques have been developed for its diagnosis. The aim of this study was to develop and evaluate three real time PCR (qPCR) protocols for different protein-coding genes (100-kDa, H and M antigens) using an animal model. Fresh and formalin-fixed and paraffin-embedded (FFPE) lung tissues from BALB/c mice inoculated i.n. with 2.5x106 Histoplasma capsulatum yeast or PBS were obtained at 1, 2, 3, 4, 8, 12 and 16 weeks post-infection. A collection of DNA from cultures representing different clades of H. capsulatum (30 strains) and other medically relevant pathogens (36 strains of related fungi and Mycobacterium tuberculosis) were used to analyze sensitivity and specificity. Analytical sensitivity and specificity were 100% when DNAs from the different strains were tested. The highest fungal burden occurred at first week post-infection and complete fungal clearance was observed after the third week; similar results were obtained when the presence of H. capsulatum yeast cells was demonstrated in histopathological analysis. In the first week post-infection, all fresh and FFPE lung tissues from H. capsulatum-infected animals were positive for the qPCR protocols tested except for the M antigen protocol, which gave variable results when fresh lung tissue samples were analyzed. In the second week, all qPCR protocols showed variable results for both fresh and FFPE tissues. Samples from the infected mice at the remaining times post-infection and uninfected mice (controls) were negative for all protocols. Good agreement was observed between CFUs, histopathological analysis and qPCR results for the 100-kDa and H antigen protocols. We successfully standardized and validated three qPCR assays for detecting H. capsulatum DNA in fresh and FFPE tissues, and conclude that the 100-kDa and H antigen molecular assays are promising tests for diagnosing this mycosis.

Amphibian populations worldwide are facing numerous threats, including the emergence and spread of infectious diseases. In the past 2 decades, Batrachochytrium dendrobatidis (Bd), a parasitic fungus, and a group of viruses comprising the genus Ranavirus have become widespread and resulted in mass mortality events and extirpations worldwide. In 2013, another novel fungus, B. salamandrivorans (Bsal), was attributed to dramatic declines in populations of fire salamander Salamandra salamandra in the Netherlands. Experimental infections demonstrated that Bsal is highly pathogenic to numerous salamander genera. In an effort to prevent the introduction of Bsal to North America, the US Fish and Wildlife Service (USFWS) listed 201 salamander species as injurious wildlife under the Lacey Act. To determine infection status and accurately assess amphibian health, the development of a sensitive and specific diagnostic assay was needed. We describe the optimization and validation of a multiplex quantitative polymerase chain reaction (qPCR) protocol for the simultaneous detection of Bd, Bsal, and frog virus 3-like ranaviruses. A synthetic genome template (gBlock®) containing the target genes from all 3 pathogens served as the positive control and allowed accurate quantification of pathogen genes. The assay was validated in the field using an established non-lethal swabbing technique to survey local amphibian populations throughout a range of habitats. This multiplex qPCR demonstrates high reproducibility, sensitivity, and was capable of detecting both Bd and ranavirus in numerous locations, species, and life stages. Bsal was not detected at any point during these sampling efforts.

Citrus melanose, caused by Diaporthe citri, is one of the most economically significant fungal diseases in China. Given that the application of fungicides is the main technique used for the control of citrus melanose, many studies have focused on enhancing the effectiveness and efficiency of the use of fungicides for the control of this economically significant disease. Here, we developed a TaqMan quantitative polymerase chain reaction (qPCR) protocol for monitoring citrus melanose via detection of suspected infected leaves and airborne conidia of D. citri. A primer pair and TaqMan probe targeting D. citri were designed to amplify a region of the fungal β-tubulin gene. The limits of detection and quantification of our TaqMan qPCR method were 159.57 and 683 copies/μL, respectively. The method detected the DNA equivalent to 47 conidia when fungal conidia were cultured in vitro. To evaluate the feasibility of our TaqMan qPCR method, symptomatic and asymptomatic leaf samples, airborne conidia samples were collected from a citrus orchard and tea garden, respectively. D. citri was detected in leaves with citrus melanose symptoms in an orchard; airborne fungal conidia of D. citri were also detected. These findings suggested that this novel TaqMan qPCR method could be used to detect D. citri both in vivo and in vitro. Our methodology could be applied as an alternative method for monitoring the changes in the incidence of D. citri in orchards.