Real-time Quantitative Polymerase Chain Reaction Method Research Articles

Pyroptosis and polarization of macrophages in septic acute lung injury induced by lipopolysaccharide in mice.

Pyroptosis and polarization are significant contributors to the onset and development of many diseases. At present, the relationship between pyroptosis and polarization in acute lung injury (ALI) caused by sepsis remains unclear. The ALI model for sepsis was created in mice and categorized into the blank control, lipopolysaccharide(LPS) group, LPS + low-dose Belnacasan group, LPS + high-dose Belnacasan group, LPS + low-dose Wedelolactone group, LPS + high-dose Wedelolactone group, and positive control group. The wet-dry specific gravity was evaluated to compare pulmonary edema. Hematoxylin-eosin, Masson, and terminal deoxynucleotidyl transferase dUTP nick end labelingstaining techniques were conducted to observe and contrast the pathological changes in lung tissue. ELISA was utilized to identify M1 and M2 macrophages and correlated inflammatory factors. Immunohistochemical staining and flow cytometry were employed to identify markers of M1 and M2 macrophages in lung tissue. Propidium iodidestaining, together with flow cytometry, was utilized to observe the degree and positive rate of pyroptosis of alveolar macrophages. Western blot analysis was conducted to detect the expression levels of Caspase 1, Caspase 11, GSDMD, and IL-18 in the lung tissues of each group. The real-time quantitative polymerase chain reactionmethod was used to ascertain relative expression levels of NLRP3, Caspase 1, Caspase 11, GSDMD, IL-18, iNOS, and Arg-1 in lung tissues of all groups. In mice with sepsis-induced ALI, both classical and nonclassical pathways of pyroptosis are observed. Inhibiting pyroptosis has been found to ameliorate lung injury, pulmonary edema, and inflammation induced by LPS. Notably, the expression of NLRP3, Caspase 1, Caspase 11, GSDMD, IL-1β, IL-18, TGF-β, CD86, CD206, iNOS, and Arg-1 were all altered in this process. Additionally, alveolar macrophages were polarized along with pyroptosis in mice with ALI caused by sepsis. Pyroptosis of alveolar macrophages in the context of ALI in mice infected with sepsis has been linked to the polarization of alveolar macrophages towardtype M1.

Imipenem resistance associated with amino acid alterations of the OprD porin in Pseudomonas aeruginosa clinical isolates.

Globally, the spread of carbapenem-resistant strains has limited treatment options for multidrug-resistant (MDR) Pseudomonas aeruginosa infections. This study aimed to determine the role of point mutations as well as the expression level of the oprD gene in the emergence of imipenem-resistant P.aeruginosa strains isolated from patients referred to Ardabil hospitals. A total of 48 imipenem-resistant clinical isolates of P. aeruginosa collected between June 2019 and January 2022 were used in this study. Detection of the oprD gene and its amino acid alterations was performed using the polymerase chain reaction (PCR) and DNA sequencing techniques. The expression level of the oprD gene in imipenem-resistant strains was determined using the real-time quantitative reverse transcription PCR (RT-PCR) method. All imipenem-resistant P. aeruginosa strains were positive for the oprD gene based on the PCR results, and also five selected isolates indicated one or more amino acid alterations. Detected amino acid alterations in the OprD porin were Ala210Ile, Gln202Glu, Ala189Val, Ala186Pro, Leu170Phe, Leu127Val, Thr115Lys, and Ser103Thr. Based on the RT-PCR results, the oprD gene was downregulated in 79.1% of imipenem-resistant P. aeruginosa strains. However, 20.9% of strains showed overexpression of the oprD gene. Probably, resistance to imipenem in these strains is associated with the presence of carbapenemases, AmpC cephalosporinase, or efflux pumps. Owing to the high prevalence of imipenem-resistant P. aeruginosa strains due to various resistance mechanisms in Ardabil hospitals, the implementation of surveillance programs to reduce the spread of these resistant microorganisms along with rational selection and prescription of antibiotics is recommended.

Methylation status, mRNA and protein expression of the SMAD4 gene in patients with non-melanocytic skin cancers.

SMAD4 is a potent tumor suppressor. SMAD4 loss increases genomic instability and plays a critical role in the DNA damage response that leads to skin cancer development. We aimed to investigate SMAD4 methylation effects on mRNA and protein expression of SMAD4 in cancer and healthy tissues from patients with basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC), and basosquamous skin cancer (BSC). The study included 17 BCC, 24 cSCC and nine BSC patients. DNA and RNA were isolated from cancerous and healthy tissues following punch biopsy. Methylation-specific polymerase chain reaction (PCR) and real-time quantitative PCR methods were used to examine SMAD4 promoter methylation and SMAD4 mRNA levels, respectively. The percentage and intensity of staining of the SMAD4 protein were determined by immunohistochemistry. The percentage of SMAD4 methylation was increased in the patients with BCC (p = 0.007), cSCC (p = 0.004), and BSC (p = 0.018) compared to the healthy tissue. SMAD4 mRNA expression was decreased in the patients with BCC (p˂0.001), cSCC (p˂0.001), and BSC (p = 0.008). The staining characteristic of SMAD4 protein was negative in the cancer tissues of the patients with cSCC (p = 0.00). Lower SMAD4 mRNA levels were observed in the poorly differentiated cSCC patients (p = 0.001). The staining characteristics of the SMAD4 protein were related to age and chronic sun exposure. Hypermethylation of SMAD4 and reduced SMAD4 mRNA expression were found to play a role in the pathogenesis of BCC, cSCC, and BSC. A decrease in SMAD4 protein expression level was observed only in cSCC patients. This suggests that epigenetic alterations to the SMAD4 gene are associated with cSCC. The name of the trial register: SMAD4 Methylation and Expression Levels in Non-melanocytic Skin Cancers; SMAD4 Protein Positivity. The registration number: NCT04759261 ( https://clinicaltrials.gov/ct2/results?term=NCT04759261 ).

Development of a Novel Real-Time Quantitative PCR Method for Detection of Ilyonectria robusta, the Predominant Species Causing Ginseng Rusty Root Rot.

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.

How we monitor the therapy effectiveness in patients with chronic myeloid leukemia - A Single center experience

Chronic myeloid leukemia (CML) is a hematological disorder characterized by increased proliferation of the granulocytic cell lineage. We diagnose CML by presence of the breakpoint cluster region-abelson (BCR-ABL1) oncogene using the quantitative real-time polymerase chain reaction (QRT-PCR). This method provides an accurate and unambiguous follow-up of the treatment response to tyrosine kinase inhibitors (TKIs). This study aimed to determine the molecular response to the first and second generation of TKIs in first and second line of treatment using the QRT-PCR method. We conducted a retrospective study on 48 CML patients treated with the first and second generation of TKIs in first and second-line treatment. Treatment responses have been followed-up every 3 months using the QRT-PCR method. Patients were divided into three groups according to molecular responses to the first line of TKIs. Results obtained in this study showed that the first group of patients did not achieve major molecular response (MMR) in the first 18 months of TKI treatment. The second and third group of patients achieved MMR and deep molecular response (DMR) in the first 18 months of TKI treatment. These results indicate that patients with MMR and DMR in the first 18 months of TKIs treatment had a favourable clinical course of the disease. Inadequate molecular responses to the first line of TKIs can be improved with in increase of the dose of TKIs or by switching to other TKIs. Continuous and timely molecular monitoring of TKI’s response in CML patients provides a careful observation of the disease's course and a proper treatment approach.

An Integrated Analytical Approach for the Characterization of Probiotic Strains in Food Supplements

Research surrounding health benefits from probiotics is becoming popular because of the increasing demand for safer products with protective and therapeutic effects. Proven benefits are species- or genus-specific; however, no certified assays are available for their characterization and quantification at the strain level in the food supplement industry. The objective of this study was to develop a strain-specific Real-time quantitative polymerase chain reaction (RT-qPCR)-based method to be implemented in routine tests for the identification and quantification of Bifidobacterium longum, Bifidobacterium animalis spp. lactis, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus helveticus, starting from a powder mixture of food supplements. The method optimization was carried out in combination with flow cytometry to compare results between the two strategies and implement the analytical workflow with the information also regarding cell viability. These assays were validated in accordance with the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) criteria using the plate count enumeration as the gold standard reference. Briefly, probiotic DNAs were extracted from two powder food supplements. Strain-specific primers targeting unique sequence regions of 16S RNA were identified and amplified by RT-qPCR. Primers were tested for specificity, sensitivity, and efficiency. Both RT-qPCR and flow-cytometry methods described in our work for the quantification and identification of Lactobacillus and Bifidobacterium strains were specific, sensitive, and precise, showing better performances with respect to the morphological colony identification. This work demonstrated that RT-qPCR can be implemented in the quality control workflow of commercial probiotic products giving more standardized and effective results regarding species discrimination.

Specific and Rapid SARS-CoV-2 Identification Based on LC-MS/MS Analysis.

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, emerged as the cause of a global crisis. Rapid and reliable clinical diagnosis is essential for effectively controlling transmission. The gold standard assay for SARS-CoV-2 identification is the highly sensitive real-time quantitative polymerase chain reaction (RT-qPCR); however, this assay depends on specialized reagents and may suffer from false results. Thus, additional assays based on different approaches could be beneficial. Here, we present a novel method for SARS-CoV-2 identification based on mass spectrometry. The approach we implemented combines a multistep procedure for the rational down-selection of a set of reliable markers out of all optional in silico derived tryptic peptides in viral proteins, followed by monitoring of peptides derived from tryptic digests of purified proteins, cell-cultured SARS-CoV-2, and nasopharyngeal (NP) swab matrix spiked with the virus. The marker selection was based on specificity to SARS-CoV-2 and on analytical parameters including sensitivity, linearity, and reproducibility. The final assay is based on six unique and specific peptide markers for SARS-CoV-2 identification. The simple and rapid (2.5 h) protocol we developed consists of virus heat inactivation and denaturation, tryptic digestion, and identification of the selected markers by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). The developed assay enabled the identification of 104 PFU/mL SARS-CoV-2 spiked into buffer. Finally, the assay was successfully applied to 16 clinical samples diagnosed by RT-qPCR, achieving 94% concordance with the current gold standard assay. To conclude, the novel MS-based assay described here is specific, rapid, simple, and is believed to provide a complementary assay to the RT-qPCR method.

Peptide Nucleic Acid (PNA)-Enhanced Specificity of a Dual-Target Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Assay for the Detection and Differentiation of SARS-CoV-2 from Related Viruses.

The threat posed by coronaviruses to human health has necessitated the development of a highly specific and sensitive viral detection method that could differentiate between the currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related coronaviruses (SARSr-CoVs). In this study, we developed a peptide nucleic acid (PNA)-based real-time quantitative polymerase chain reaction (RT-qPCR) assay targeting the N gene to efficiently discriminate SARS-CoV-2 from other SARSr-CoVs in human clinical samples. Without compromising the sensitivity, this method significantly enhanced the specificity of SARS-CoV-2 detection by 100-fold as compared to conventional RT-qPCR. In addition, we designed an RT-qPCR method for the sensitive and universal detection of ORF3ab-E genes of SARSr-CoV with a limit of detection (LOD) of 3.3 RNA copies per microliter. Thus, the developed assay serves as a confirmative dual-target detection method. Our PNA-mediated dual-target RT-qPCR assay can detect clinical SARS-CoV-2 samples in the range of 18.10–35.19 Ct values with an 82.6–100% detection rate. Furthermore, our assay showed no cross-reactions with other coronaviruses such as human coronaviruses (229E, NL63, and OC43) and Middle East respiratory syndrome coronavirus, influenza viruses (Type B, H1N1, H3N2, HPAI H5Nx, and H7N9), and other respiratory disease-causing viruses (MPV, RSV A, RSV B, PIV, AdV, and HRV). We, thus, developed a PNA-based RT-qPCR assay that differentiates emerging pathogens such as SARS-CoV-2 from closely related viruses such as SARSr-CoV and allows diagnosis of infections related to already identified or new coronavirus strains.

In silico assessment of the impact of 2019 novel coronavirus genomic variation on the efficiency of published real-time quantitative polymerase chain reaction detection assays.

To the Editor: In December 2019, coronavirus disease 2019 outbreak caused by the 2019 novel coronavirus (2019-nCoV) happened in Wuhan, China. Now, it has posed a worldwide public health threat. Real-time quantitative polymerase chain reaction (RT-qPCR) was recommended as an effective pathogen detection method and has played an important role in prevention and control of the current outbreak. Many research institutions have released their primer sets for RT-qPCR. If the variant sites were located in the primer regions, the efficiency of RT-qPCR would be reduced, thus possibly causing false negative results, and leading to unpredictable impact on the diagnosis of patients and the control of this outbreak. Therefore, a comprehensive investigation on 2019-nCoV genome variation is necessary to evaluate the effectiveness of current released RT-qPCR methods. Here, we analyzed 77 public full-length genome sequences of 2019-nCoV from the GISAID website [Supplementary Table 1, https://links.lww.com/CM9/A222]. All the sequences were aligned by using MAFFT v7.450.[1] A total of 85 variant sites were found, all of which are single nucleotide variants. Among the 85 variant sites, seven were shared by two 2019-nCoV sequences and nine were found in three or more sequences [Supplementary Table 2, https://links.lww.com/CM9/A223]. We investigated the published 2019-nCoV RT-qPCR detection assays and found a total number of 13 RT-qPCR primer sets designed by eight institutions [Table 1, Supplementary Table 3, https://links.lww.com/CM9/A224]. These primers were designed to amplify genes of ORF1ab, Spike (S), Envelope (E), and Nucleocapsid (N). The reverse primers of primer sets 3 and 6 had one mismatch against all of the released 2019-nCoV sequences. Among all the observed variation sites, three (positions on the reference genome IVDC-HB-01: 28291, 28688, and 29200) are separately located on the region of forward primers of the primer sets 7 and 9, and the probe of the primer set 8. It is noted that the above three variations was found in the sequences BetaCoV/Shenzhen/SZTH-003/2020, BetaCoV/Shandong/IVDC-SD-001/2020|EPI_ISL_408482, and BetaCoV/Chongqing/YC01/2020|EPI_ISL_408478, respectively. These variants may affect the RT-qPCR detection efficiency. In particular, variations on the probe region of the primer set 8 may have largely negative effects on detection efficiency according to the previous research.[2]Table 1: The detailed information of publicly released 2019-nCoV RT-qPCR primers.In conclusion, using any of the five RT-qPCR primer sets mentioned above to detect 2019-nCoV may potentially cause false-negative results. Among the five, two have mismatches and three contain some 2019-nCoV genome variants which occurred during the outbreak. It is worth noting that the three primer sets containing variants are all located on the N gene. Therefore, it is suggested that conservative regions, such as nsp12 (RdRp) gene, would be preferable primer targets. Although the multiple-targets designation of RT-qPCR protocol would reduce the false-negative results caused by genome variation, more careful performance evaluation of the currently used primers is needed. Moreover, it is necessary to keep continuous surveillance on the genome variants and their effects on the RT-qPCR assays during the whole outbreak. Acknowledgements We gratefully acknowledge the Authors, the Originating and Submitting Laboratories for their sequences and metadata shared through GISAID, on which this research is based. All submitters of data may be contacted directly via https://www.gisaid.org. Funding This work was supported by the National Key Research & Development Program of China (No. 2020YFC0840900) and the Beijing Municipal Science and Technology Project (No. Z201100001020004). Conflicts of interest None.

Viral-bacterial (co-)occurrence in the upper airways and the risk of childhood pneumonia in resource-limited settings

ObjectiveTo examine the association between bacterial-viral co-occurrence in the nasopharynx and the risk of community acquired pneumonia (CAP) in young children living in resource-limited settings. MethodsA case-control study was conducted between January and December 2017 in Moshi, Tanzania. Children 2–59 months with CAP and healthy controls were enrolled. RSV and Influenza A/B were detected with a standardized polymerase chain reaction (PCR) method, and a simplified real-time quantitative PCR method, without sample pre-processing, was developed to detect bacterial pathogens in nasopharyngeal samples. ResultsA total of 109 CAP patients and 324 healthy controls were enrolled. Co-detection of H. influenzae and S. pneumoniae in nasopharyngeal swabs was linked with higher odds of CAP (aOR=3.2, 95% CI=1.1–9.5). The majority of the H. influenzae isolated in cases and controls (95.8%) were non-typeable. Of the viruses examined, respiratory syncytial virus (RSV) was most common (n = 31, 7.2%) in cases and controls. Children with RSV had 8.4 times higher odds to develop pneumonia than healthy children (aOR=8.4, 95%CI= 3.2 – 22.1). ConclusionsCo-occurence of H. influenzae and S. pneumoniae in the nasopharynx was strongly associated with CAP. The high prevalence of non-typeable H. influenzae might be a sign of replacement as a consequence of Haemophilus influenzae type b vaccination.

Rapid and sensitive detection of Shigella flexneri using fluorescent microspheres as label for immunochromatographic test strip.

Bacillary dysentery caused by Shigella genus is a major cause of morbidity and mortality worldwide. In China, the popular strain was mainly Shigella flexneri (S. flexneri). Therefore, fluorescent microspheres (FMs)-based immunochromatographic test strip (ICTS), as a novel, reliable, sensitive and uncomplicated method, was evaluated to detect S. flexneri. Sixty-three clinical samples of S. flexneri were collected in this paper. Polymerase chain reaction (PCR) combined with FMs-ICTS based on magnetic purification assay was developed for the quantitative detection of Shigella. And the genus-specific gene of ipaH and drug resistant gene of CTX-M-9 from Shigella were selected to investigate the potential of this new method. The sensitivity and specificity of this method were demonstrated by classical microbiological methods (API Coryne System), PCR assay based on agarose gel electrophoresis (PCR-GE) and the real-time fluorescent quantitative PCR (RTFQ-PCR) method. Under optimized conditions, the lower detection limits of PCR-ICTS, PCR-GE and RTFQ-PCR were 2.5×10-7, 2.5×10-5 and the 3.2×10-7 ng/µL, respectively. Experiments demonstrated the PCR-ICTS has a diagnostic agreement of 100% with conventional PCR and RTFQ-PCR on detection of clinical samples and could correctly recognize Shigella and non-Shigella from different microbial samples. After the purification of PCR products with Silicon coated magnetic nanoparticles (Si-MNPs), the false positive results were removed because of the strong screening ability of the purification process. Our results showed that FM-based ICTS was promising for measurable and sensitive detection of S. flexneri within 3 h. The results from immunochromatographic test were agreement with those from API Coryne system and RTFQ-PCR. Hence, this developed method might be useful for screening and monitoring clinical sample of S. flexneri, due to its speed, non-poisonous, simplicity and low-cost and helpful for promoting the prevention and control of communicable diseases caused by enteric pathogens such as S. flexneri.

A new highly sensitive real-time quantitative-PCR method for detection of BCR-ABL1 to monitor minimal residual disease in chronic myeloid leukemia after discontinuation of imatinib.

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 fusion protein, encoded by the Philadelphia chromosome, have drastically improved the outcomes for patients with chronic myeloid leukemia (CML). Although several real-time quantitative polymerase chain reaction (RQ-PCR) kits for the detection of BCR-ABL1 transcripts are commercially available, their accuracy and efficiency in laboratory practice require reevaluation. We have developed a new in-house RQ-PCR method to detect minimal residual disease (MRD) in CML cases. MRD was analyzed in 102 patients with CML from the DOMEST study, a clinical trial to study the rationale for imatinib mesylate discontinuation in Japan. The BCR-ABL1/ABL1 ratio was evaluated using the international standard (IS) ratio, where IS < 0.1% was defined as a major molecular response. At enrollment, BCR-ABL1 transcripts were undetectable in all samples using a widely-applied RQ-PCR method performed in the commercial laboratory, BML (BML Inc., Tokyo, Japan); however, the in-house method detected the BCR-ABL1 transcripts in five samples (5%) (mean IS ratio: 0.0062 ± 0.0010%). After discontinuation of imatinib, BCR-ABL1 transcripts were detected using the in-house RQ-PCR in 21 patients (21%) that were not positive using the BML method. Nineteen samples were also tested using a commercially available RQ-PCR assay kit with a detection limit of IS ratio, 0.0032 (ODK-1201, Otsuka Pharmaceutical Co., Tokyo, Japan). This method detected low levels of BCR-ABL1 transcripts in 14 samples (74%), but scored negative for five samples (26%) that were positive using the in-house method. From the perspective of the in-house RQ-PCR method, number of patients confirmed loss of MMR was 4. These data suggest that our new in-house RQ-PCR method is effective for monitoring MRD in CML.

Feasibility of amplification refractory mutation system in fast detection of clarithromycin resistance of Helicobacter pylori in gastric mucosa

Objective To evaluate the feasibility of the combination of amplification refractory mutation system (ARMS) and quantitative real-time polymerase chain reaction (PCR) method in fast detection of clarithromycin resistance of Helicobacter pylori (H.pylori) in gastric mucosa. Methods A total of 150 gastric mucosal specimens with positive H. pylori culture were collected from the H. pylori positive patients who failed in H. pylori eradication from January to August in 2013. The drug resistant gene mutation types of H. pylori in these samples were detected by quantitative real-time PCR based on ARMS. And the accuracy was confirmed by sequencing. The clarithromycin resistance of H. pylori was determined by E-assay. Chi-square test was used for statistical analysis. Results Among 149 gastric mucosal specimens (one specimens without wild type or mutation type had been eliminated), the results of quantitative real-time PCR based on ARMS of two samples were not consistent with the results of sequencing; the consistent rate was 98.7% (147/149). Among 149 specimens with positive H. pylori culture, 104 samples (69.8%) were clarithromycin resistance. In 101 samples the clarithromycin resistance was detected by quantitative real-time PCR based on ARMS; the consistent rate was 97.1% (101/104). Both E-assay and clarithromycin resistant rate detected by E-assay or quantitative real-time PCR based on ARMS was 69.8% (104/149) and 67.8% (101/149), respectively, and the difference was not significant (χ2=0.141, P=0.932). Conclusion The combination of ARMS and quantitative real-time PCR method in fast detection of clarithromycin resistance of H. pylori in gastric mucosa is strongly feasible and highly consistent has high consistent rate with sequencing and E-assay. Key words: Helicobacter pylori; Amplification refractory mutation system; Mutation; Resistance

Utility of Real-Time Quantitative Polymerase Chain Reaction in Detecting Mycobacterium tuberculosis.

This study aimed to assess the value of real-time quantitative polymerase chain reaction (RT-qPCR) for the detection of Mycobacterium tuberculosis (MTB). Samples from 192 patients with suspected MTB were examined by RT-qPCR and an improved Löwenstein–Jensen (L-J) culture method. To evaluate the diagnostic usefulness of RT-qPCR in detecting MTB, a receiver operating characteristic (ROC) curve for RT-qPCR was generated, and the area under the curve (AUC) as well as a cutoff value was calculated. Using the L-J culture method as the gold standard, accuracy of the RT-qPCR method for detecting MTB was 92.7%, with sensitivity and specificity of 62.5% and 97.02%, respectively. In comparison with the improved L-J culture method, the AUC of RT-qPCR ROC curve was 0.957, which was statistically significant (p < 0.001). The Youden Index reached the maximum value (0.88) for gene copy number of 794.5 IU/mL, which was used as the cutoff value. RT-qPCR detection of MTB yielded results consistent with those of the improved L-J culture method, with high accuracy. RT-qPCR may be used as an auxiliary method for etiological diagnosis of tuberculosis.

Droplet-Based Multivolume Digital Polymerase Chain Reaction by a Surface-Assisted Multifactor Fluid Segmentation Approach

Here we developed the surface-assisted multifactor fluid segmentation (SAMFS), an automated, fast, and flexible approach for generating a two-dimensional droplet array with tunable droplet volumes, for multivolume digital polymerase chain reaction (PCR). The SAMFS was developed based on the combination of robotic liquid handling and surface-assisted droplet generation techniques, where a continuous aqueous stream that flowed out from a capillary probe was segmented and immobilized on hydrophilic micropillars of a microchip into massive oil-covered droplets with the probe rapidly scanning over the microchip. We studied various factors affecting the droplet generation process, including micropillar top area, distance between adjacent micropillars, aqueous stream flow rate, and microchip moving speed, and demonstrated a high droplet generation throughput up to 50 droplets/s and a largest droplet volume adjusting range from 0.25 to 350 nL. The SAMFS approach was adopted to form an oil-covered array of 994 droplets with four different volumes (1.2, 6, 30, and 150 nL droplets) required for multivolume digital PCR within 8 min. The droplet array system was applied in absolute quantification of plasmid DNA under the multivolume digital PCR mode with a dynamic range spanning 4 orders of magnitude, as well as measurement of HER2 expression levels in different breast cancer cell lines. The results are consistent to those obtained by quantitative real-time PCR method, while the present one has higher precision.

A new specific reference gene based on growth hormone gene (GH1) used for detection and relative quantification of Aquadvantage® GM salmon (Salmo salar L.) in food products

Genetic transformation of fish is mainly oriented towards the improvement of growth for the benefit of the aquaculture. Actually, Atlantic salmon (Salmo salar) is the species most transformed to achieve growth rates quite large compared to the wild. To anticipate the presence of contaminations with GM salmon in fish markets and the lack of labeling regulations with a mandatory threshold, the proper methods are needed to test the authenticity of the ingredients. A quantitative real-time polymerase chain reaction (QRT-PCR) method was used in this study. Ct values were obtained and validated using 15 processed food containing salmon. The relative and absolute limits of detection were 0.01% and 0.01ng/μl of genomic DNA, respectively. Results demonstrate that the developed QRT-PCR method is suitable specifically for identification of S. salar in food ingredients based on the salmon growth hormone gene 1 (GH1). The processes used to develop the specific salmon reference gene case study are intended to serve as a model for performing quantification of Aquadvantage® GM salmon on future genetically modified (GM) fish to be commercialized.

Development and Efficacy of Real-Time PCR in the Diagnosis of Vivax Malaria Using Field Samples in the Republic of Korea

The development of sensitive, rapid, and accurate diagnostic methods for vivax malaria is essential for the effective control of malaria in the Republic of Korea, where vivax malaria patients usually show low parasitemia. In this study, a TaqMan-based real-time polymerase chain reaction (PCR) method was established and compared with other PCR-based assays, including nested PCR, loop-mediated isothermal amplification, and multiplex PCR, using samples from febrile patients with suspected vivax malaria. The established real-time PCR had a high sensitivity (99.6%) and specificity (100%). Therefore, this sensitive, specific, rapid, and quantitative real-time PCR method could be used for the routine diagnosis of vivax malaria in the laboratory of the Korea National Institute of Health.

Effect of paddy-upland rotation on methanogenic archaeal community structure in paddy field soil.

Methanogenic archaea are strict anaerobes and demand highly reduced conditions to produce methane in paddy field soil. However, methanogenic archaea survive well under upland and aerated conditions in paddy fields and exhibit stable community. In the present study, methanogenic archaeal community was investigated in fields where paddy rice (Oryza sativa L.) under flooded conditions was rotated with soybean (Glycine max [L.] Merr.) under upland conditions at different rotation histories, by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR methods targeting 16S rRNA and mcrA genes, respectively. Soil samples collected from the fields before flooding or seeding, during crop cultivation and after harvest of crops were analyzed. The abundance of the methanogenic archaeal populations decreased to about one-tenth in the rotational plots than in the consecutive paddy (control) plots. The composition of the methanogenic archaeal community also changed. Most members of the methanogenic archaea consisting of the orders Methanosarcinales, Methanocellales, Methanomicrobiales, and Methanobacteriales existed autochthonously in both the control and rotational plots, while some were strongly affected in the rotational plots, with fatal effect to some members belonging to the Methanosarcinales. This study revealed that the upland conversion for one or longer than 1 year in the rotational system affected the methanogenic archaeal community structure and was fatal to some members of methanogenic archaea in paddy field soil.

Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet “Beta vulgaris L.”: GMO application

KEY MESSAGE : Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis. The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R (2)>0.994) over sugar beet standard concentrations ranging from 20,000 to 10HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.

Quantification of Epstein-Barr Virus DNA Is Helpful for Evaluation of Chronic Active Epstein-Barr Virus Infection

Chronic active Epstein-Barr virus infection (CAEBV) presents with chronic or recurrent infectious mononucleosis-like symptoms, such as low-grade fever, liver dysfunction, lymphadenopathy, and hepatosplenomegaly. Immunological methods are useful for the diagnosis of viral infections. However, CAEBV patients do not necessarily have high titers of Epstein-Barr virus (EBV)-specific antibodies. Hosts that are immunocompromised after hematopoietic stem cell transplantations sometimes suffer from systemic EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and EBV-positive lymphoma. Patients with EBV-associated diseases are often diagnosed by analyses of bone marrow. Cytomegalovirus (CMV) can cause serious pneumonia or retinitis in immunocompromised hosts. In order to noninvasively understand the clinical status of patients with EBV-associated diseases, we conducted real-time polymerase chain reaction (PCR) methods in their peripheral blood in order to quantify EBV and CMV DNA levels, which reflect viral activity. Here, we describe a 30-year-old Japanese female patient with CAEBV. The patient had repeated fever, fatigue, and liver dysfunction. The histopathological results of liver biopsies were positive for EBV-encoded RNA-1. Acute hepatitis was associated with the EBV infection. The whole-blood EBV DNA levels were high and above 1.0 × 10⁷ copies/mL. After immunosuppressive and antiviral therapies, EBV DNA levels lowered. However, she had to receive bone marrow transplantation because of her EBV-HLH. As the number of lymphocytes increased in the post-transplantation period, EBV DNA levels gradually increased again. The simultaneous detection of CMV DNA was more sensitive than the CMV antigenemia test that is often used to diagnose CMV infections. Unfortunately, the patient died due to a fungal infection. Observing EBV DNA levels closely with real-time quantitative PCR methods is helpful for evaluating the changes in the clinical course.