No abstract available.
Real-Time Polymerase Chain Reaction*

The aim of this study was to investigate the pattern of distribution of mating type (MAT) genes of Tuber indicum in ectomycorhizosphere soils from natural T. indicum-producing areas and cultivated truffle orchards and ascocarp samples from different regions. Quantitative real-time PCR and multiplex PCR were used to weight the copy numbers of MAT1-1-1 and MAT1-2-1 in natural truffle soils and cultivated orchard soils. The effect of limestone on the pattern of truffle MAT genes and the correlation between soil properties and the proportion of MAT genes were also assessed. These results indicated that an uneven and nonrandom distribution of MAT genes was common in truffle-producing areas, cultivated truffle orchards, and ascocarps gleba. The competition between the two mating type genes and the expansion of unbalanced distribution was found to be closely related to truffle fructification. Limestone treatments failed to alter the proportion of the two mating type genes in the soil. The content of available phosphorus in soil was significantly correlated with the value of MAT1-1-1/MAT1-2-1 in cultivated and natural ectomycorhizosphere soils. The application of real-time quantitative PCR can provide reference for monitoring the dynamic changes of mating type genes in soil. This study investigates the distributional pattern of T. indicum MAT genes in the ectomycorhizosphere soil and ascocarp gleba from different regions, which may provide a foundation for the cultivation of T. indicum.
Calcium Carbonate ; Multiplex Polymerase Chain Reaction ; Phosphorus ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Soil

BACKGROUND: We comparatively evaluated the performance of the conventional COBAS Amplicor HCV test v2.0 (CAM; Roche Molecular Systems, USA) and the newly developed COBAS AmpliPrep/COBAS TaqMan HCV test v2.0 (CAP/CTM; Roche Molecular Systems) for qualitative detection of hepatitis C virus (HCV) RNA in clinical samples. METHODS: Six hundred serum samples (100 HCV-positive, 500 HCV-negative, as determined by CAM) were selected and analysed using the new qualitative HCV RNA test, CAP/CTM qualitative test. Results were compared by confirmatory CAP/CTM quantitative test, which is a quantitative HCV RNA real-time polymerase chain reaction by Roche Molecular Systems, and anti-HCV test (Roche Diagnostics GmbH, Germany). Twenty-two additional serum samples, which gave a gray zone result by CAM, were selected for comparison. RESULTS: The two qualitative HCV RNA assays yielded concordant results for 586 of 600 tested samples (concordance rate, 97.7%; kappa coefficient, 0.92; 95% confidence interval [CI], 0.87 to 0.96; P<0.001). Upon re-testing by CAM, we found that the concordance rate increased to 98.2% (kappa coefficient, 0.93; 95% CI, 0.89 to 0.97; P<0.001). The additional 22 samples showing gray zone results for CAM were retested and were also tested by CAP/CTM. The results for 13 of these samples changed to negative and were now concordant with the CAP/CTM and confirmatory CAP/CTM quantitative results. For the remaining samples, the results were variable. For all the 22 samples, the results of the new CAP/CTM were in agreement with those obtained by confirmatory CAP/CTM quantitative test. CONCLUSIONS: The results of the two assays were in good agreement, with 97.7% concordance rate. However, CAP/CTM is more sensitive than CAM and showed no gray zone results. Therefore, it can be a more efficient and useful test for the qualitative detection of HCV RNA in clinical samples.
Hepacivirus* ; Hepatitis C* ; Hepatitis* ; Real-Time Polymerase Chain Reaction ; RNA

To identify herpesviral virions secreted by viral replication, we have established the nuclease-resistance assay (NRA) as a comparative analysis to other conventional assays, including electron microscopy (EM). For this study, we used an efficient experimental in vitro infection model for Alcelaphine herpesvirus 1 (AlHV-1), a gamma herpesvirus, to propagate the virus. The NRA could identify extracellular, cell-free, enveloped virions in the supernatants after 24 hours post inoculation (h.p.i.) of AlHV-1. The results of EM observation were correlated with those of NRA. Mature virions were observed in the clarified, concentrated supernatants from 24 h.p.i. by EM. These results show that sensitivity of the NRA is comparable with that of EM for the identification of mature enveloped virions, which directly presents evidence of herpesviral lytic replication. NRA allows us to differentiate the virus from other member of Herpesviridae, and has extended the possibility of analysis for quantification of shedding viruses when used in conjunction with real-time polymerase chain reaction (PCR).
Herpesviridae ; Microscopy, Electron* ; Real-Time Polymerase Chain Reaction ; Virion*

BACKGROUND: Broth cultures are increasingly used to detect acid-fast bacilli (AFB). Rapid, simple and accurate methods for differentiation of Mycobacterium tuberculosis complex (MTBC) and nontuberculosis mycobacteria from broth cultures are needed. Immunochromatographic assays (ICTs) for identification of MTBC have been developed. METHODS: The abilities of the BD MGIT TBc Identification Test (Becton Dickinson, USA) and the SD Bioline TB Ag MPT64 (Standard Diagnostics, Korea) to detect MTBC were evaluated in 44 AFB-positive broth cultures. The results of 2 ICTs were compared to those of real-time PCR. RESULTS: The BD MGIT TBc Identification Test and the SD Bioline TB Ag MPT64 showed concordant results with real-time PCR by 100% and 97.7%, respectively. The sensitivity of the BD MGIT TBc Identification and the SD Bioline TB Ag MPT64 was 100% for both, and the specificities of those were 100% and 95.2%, respectively. CONCLUSIONS: Both ICTs are rapid methods for identification of MTBC from broth cultures, and the results of ICTs are in accord with those of real-time PCR.
Immunochromatography ; Mycobacterium ; Mycobacterium tuberculosis ; Real-Time Polymerase Chain Reaction

For our survey of the infection frequency and mixed infection of the viruses causing acute respiratory syndromes, we analyzed those viruses from acute respiratory patients in Seoul. Total 1,038 specimens of oropharyngeal swab were tested by the real-time polymerase chain reaction (PCR) kit (Kogenebiotech, Korea) from Jan. to Dec. in 2013. Virus detection rate causing acute respiratory infection was 46% (476/1,038). The most frequently isolated virus was only hRV (21.6%, 103/476), followed by only ADV (8.96%, 93/476), only IFV A (H3N2) (18.1%, 86/476), and only hCoV (7.8%, 37/476) etc. Most of acute respiratory viruses had severe fever. Infection frequency information and mixed infection status on respiratory viruses circulating in Seoul will be helpful for the management of acute respiratory infection and for epidemiological continuous studies.
Coinfection* ; Fever ; Humans ; Real-Time Polymerase Chain Reaction ; Seoul

No abstract available.
Diagnosis* ; Real-Time Polymerase Chain Reaction* ; Tuberculosis, Pulmonary*

Tree shrew is a novel and high-quality experimental animal model. In this study, the real-time polymerase chain reaction methods were established to detect infection-related cytokines interleukin-6 (IL-6), IL-8, IL-10, IL-17A, interferon-γ (IFN-γ) and housekeeping gene glyceraldehyde-phosphate dehydrogenase ( ) of tree shrew. The results indicated that the establised methods had good specificity. The high point of the linear range of these reagents reached 1 × 10 copies, and the low points ranged from 10 copies (IL-6, IL-17A), 100 copies (IL-10, ) to 1 000 copies (IL-8, IFN-γ). In this interval, the linear correlation coefficient of each reagent was greater than 0.99. The lowest detectable values of IL-6, IL-8, IL-10, IL-17A, IFN-γ and were 8, 8, 4, 8, 128 and 4 copies, respectively. The results showed that the established detection methods had good specificity, sensitivity and wide linear range. The methods were suitable for detection of multiple concentration range samples, and could be used for the subsequent studies of tree shrew cytokines.
Animals ; Cytokines ; analysis ; Real-Time Polymerase Chain Reaction ; Shrews

The specific PCR primer was designed base on ITS2 sequence in GenBank, and we developed a SYBRGreen real-time fluorescence quantitative PCR system for identification of Crocus sativus and Carthamus tinctorius source. Compared with Chinese herbal medicine DNA barcode technique, this method showed characteristics of shorter time, higher specificity and sensitivity. Using this method to detect 15 samples, 4 were C. sativus, 8 were C. tinctorius, and the other 3 samples were none of them. The result was in accordance with Chinese herbal medicine DNA barcode. This study lays the foundation for identification of related Chinese medical materials.
Carthamus tinctorius ; Crocus ; Real-Time Polymerase Chain Reaction

OBJECTIVE: To establish and modify quantitative real-time polymerase chain reaction (qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes. METHODS: A database of capsular polysaccharide ( cps) loci sequences was generated, covering 97 pneumococcal serotypes. Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs. A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR, while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay. A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping. RESULTS: A total of 97 pneumococcal serotyping assays based on qPCR were established and modified, which included 64 serotypes previously reported as well as an additional 33 serotypes. Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system. A total of 97 pneumococcal serotypes, which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups, could not be identified as individual serotypes. The sensitivity of qPCR assays based on 27 target sequences was 1-100 copies/µL. The specificity of the qPCR assays was 100%, which were tested by a panel of 90 serotypes of the pneumococcal reference strains. CONCLUSION A total of 27 novel qPCR assays were established and modified to analyze 97 pneumococcal serotypes.
Real-Time Polymerase Chain Reaction ; Serotyping ; Streptococcus pneumoniae/genetics* ; Serogroup