Guanidine Isothiocyanate Research Articles

Ammonium sulfate denatures transport medium less dependent on guanidinium isothiocyanate and enables SARS-CoV-2 RNA and antigen detection compatibility.

Rapid identification of infected individuals through viral RNA or antigen detection followed by effective personal isolation is usually the most effective way to prevent the spread of a newly emerging virus. Large-scale detection involves mass specimen collection and transportation. For biosafety reasons, denaturing viral transport medium has been extensively used during the SARS-CoV-2 pandemic. However, the high concentrations of guanidinium isothiocyanate (GITC) in such media have raised issues around sufficient GITC supply and laboratory safety. Moreover, there is a lack of denaturing transport media compatible with SARS-CoV-2 RNA and antigen detection. Here, we tested whether supplementing media containing low concentrations of GITC with ammonium sulfate (AS) would affect the throat-swab detection of SARS-CoV-2 or a viral inactivation assay targeting coronavirus and other enveloped and non-enveloped viruses. The effect of adding AS to the media on RNA stability and its compatibility with SARS-CoV-2 antigen detection were also tested. We found that adding AS to the denaturing transport media reduced the need for high levels of GITC, improved SARS-COV-2 RNA detection without compromising virus inactivation, and enabled the denaturing transport media compatible with SARS-CoV-2 antigen detection.

Collection and transportation of SARS-CoV-2 and influenza A virus diagnostic samples: Optimizing the usage of guanidine-based chaotropic salts for enhanced biosafety and viral genome preservation

Many virus lysis/transport buffers used in molecular diagnostics, including the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, contain guanidine-based chaotropic salts, primarily guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC). Although the virucidal effects of GuHCl and GITC alone against some enveloped viruses have been established, standardized data on their optimum virucidal concentrations against SARS-CoV-2 and effects on viral RNA stability are scarce. Thus, we aimed to determine the optimum virucidal concentrations of GuHCl and GITC against SARS-CoV-2 compared to influenza A virus (IAV), another enveloped respiratory virus. We also evaluated the effectiveness of viral RNA stabilization at the determined optimum virucidal concentrations under high-temperature conditions (35°C) using virus-specific real-time reverse transcription polymerase chain reaction. Both viruses were potently inactivated by 1.0 M GITC and 2.5 M GuHCl, but the GuHCl concentration for efficient SARS-CoV-2 inactivation was slightly higher than that for IAV inactivation. GITC showed better viral RNA stability than GuHCl at the optimum virucidal concentrations. An increased concentration of GuHCl or GITC increased viral RNA degradation at 35°C. Our findings highlight the need to standardize GuHCl and GITC concentrations in virus lysis/transport buffers and the potential application of these guanidine-based salts alone as virus inactivation solutions in SARS-CoV-2 and IAV molecular diagnostics.

A Comprehensive Comparison of Rapid RNA Extraction Methods for Detection of SARS-CoV-2 as the Infectious Agent of the Upper Respiratory Tract using Direct RT-LAMP Assay.

The current COVID-19 pandemic has highlighted the need for faster and more cost-effective diagnostic methods. The RNA extraction step in current diagnostic methods, such as real-time qPCR, increases the cost and time required for testing. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is a promising technique for developing diagnostic tests with desired sensitivity and specificity without the need for RNA extraction. An RT-LAMP assay was developed to detect SARS-CoV-2 with a sensitivity of 0.5 copies of positive control plasmid per microliter in 40 min. Several rapid RNA extraction protocols were evaluated using different reagents, including bovine serum albumin, Triton X-100, Tween 20, proteinase K, guanidine hydrochloride, guanidinium isothiocyanate (GITC), and thermal treatment. Finally, the sensitivity and specificity of the developed direct RT-LAMP were determined using 150 upper respiratory tract samples. Method 10 was selected as the most efficient protocol for the RNA extraction step. The sensitivity and specificity of the developed direct RT-LAMP assay with clinical samples were estimated at 98.4% and 88.8%, respectively. These results suggest that the combination of GITC and Triton X-100 detergent is a highly efficient method for RNA extraction and direct RT-LAMP detection of SARS-CoV-2 in clinical samples, providing a valuable tool for the rapid and cost-effective diagnosis of COVID-19.

How to better select SARS-CoV-2 preservation solution of virus nucleic acid testing.

Sampling and testing for SARS-CoV-2 is a widely recognized method for identifying patients with COVID-19. However, there is limited research available on the stability of nucleic acids in viral storage solutions. This paper investigates the components that provide better protection for virus and nucleic acid detection. The study utilized real-time quantitative fluorescent PCR to detect SARS-CoV-2 and evaluate the preservation effect and stability of SARS-CoV-2 viral storage solution under various conditions, including different guanidinium salts, brands, and storage conditions. All brands of inactivated virus preservation solutions demonstrated effective preservation and stability. However, 0.5 mol/L guanidine hydrochloride and guanidine isothiocyanate solutions exhibited poor antiseptic effects. Additionally, refrigerated storage showed better preservation compared to room temperature storage. We recommend using inactivated virus collection solution to preserve and transport samples and testing preferably within 6 hours to reduce false negatives of NAT results.

Cell-free plasma miRNAs analysis for low invasive lung cancer diagnostics

Introduction. The high mortality rate in patients with lung cancer (LC) is due to the lack of highly sensitive diagnostic markers of this disease. Genetic and epigenetic alterations in tumor cells, for example, aberrant microRNA expression, can be proposed. It is known that extracellular/circulating microRNA of biological fluids, in complexes with proteins, or packaged in extracellular vesicles is of interest for the diagnosis of tumor diseases.Aim. To perform a comparative analysis of miRNA expression in plasma and plasma extracellular vesicles of LC patients and healthy donors. Based on the obtained results, to propose a diagnostic panel to identify patients with LC.Materials and methods. Blood plasma was obtained from blood samples of healthy donors and LC patients by sequential centrifugation. Then, a fraction of extracellular vesicles (40–150 nm in size) was isolated from a part of the obtained plasma supernatant by the method of aggregation-precipitation with polyethylene glycol/blue dextran. MicroRNAs were isolated from both blood plasma fractions of patients and healthy donors using guanidine isothiocyanate and octanoic acid. Expression of 17 miRNAs most characteristic for the development of LC according to our and literature data in the above-mentioned blood plasma fractions was analyzed by stem-loop reverse transcription polymerase chain reaction.Results. 29 and 10 miRNA pairs were differentially expressed in plasma extracellular vesicles and plasma of lung cancer patients and donors. Thus, plasma extracellular vesicles are characterized by greater potential as a source for miRNA based lung cancer diagnostic panels in comparison with blood plasma. Diagnostic algorithm based on aberrant miRNA expression of 8 different miRNAs (miRNA-30e, -1, -125b, -133, -222, -374, -425, -660) composed in 6 pairs was designed. This algorithm allows to diagnose 100 % of patients with lung cancer stages II–IV.Conclusion. Extracellular plasma vesicles represent a promising source of diagnostically significant microRNAs compared to plasma microRNAs. For the diagnosis of patients with non-small cell lung cancer with 100 % sensitivity and specificity, a panel of 8 microRNAs (6 miRNA pairs) was proposed.

Prognostic importance of PD-L1 expression in bladder cancer patients.

PD-L1/PD-1 molecules are known as important mediators in immune-escape mechanisms of tumors. PD-L1 is highly expressed in various malignancies, including bladder cancer. However, the prognostic value of PD-L1 in bladder cancer patients remains controversial. To investigate the prognostic significance of PD-L1 expression in tumor tissues of bladder cancer patients. RNA was isolated from FFPE tumor tissues of 48 bladder cancer patients using the monophasic phenol and guanidine isothiocyanate method. Total RNA was converted to cDNA and gene expression levels were analyzed by qRT-PCR. The differential expression levels of the PD-L1 gene between tumor grade and cancer stage groups were analyzed by independent student's t-test and one-way ANOVA. Statistically significantly increased PD-L1 expression was observed in the high-grade tumor group (p < 0.05). No significant difference in PD-L1 expression was found among pTa, pT1, and pT2 groups. In addition, the difference in overall survival was not significantly different between groups. The results showed that high PD-L1 expression in bladder cancer was associated with tumor aggressiveness and grade. Despite the inability of the qRT-PCR to show the PD-L1 expression at different locations of tumor tissue, evaluation of PD-L1 mRNA expression by qRT-PCR, which is a highly sensitive and specific assay, appears to be a robust approach. Furthermore, these findings may contribute to a rationale for recommending anti-PD-L1 immunotherapy as an alternative to standard therapy for bladder cancer patients who are most likely to benefit from it.

Toxic effects of trace phenol/guanidine isothiocyanate (P/GI) on cells cultured nearby in covered 96-well plates

BackgroundA mixture of phenol and guanidine isothiocyanate (“P/GI”, the principal components of TRIzol™ and similar products) is routinely used to isolate RNA, DNA, and proteins from a single specimen. In time-course experiments of cells grown in tissue culture, replicate wells are often harvested sequentially and compared, with the assumption that in-well lysis and complete aspiration of P/GI has no effect on continuing cultures in nearby wells.MethodsTo test this assumption, we investigated morphology and function of RAW 264.7 cells (an immortalized mouse macrophage cell line) cultured in covered 96-well plates for 4, 8, or 24 h at varying distances from a single control well or a well into which P/GI had been deposited and immediately aspirated completely.ResultsTime- and distance-dependent disruptions resulting from proximity to a single well containing trace residual P/GI were seen in cell morphology (blebbing, cytoplasmic disruption, and accumulation of intracellular vesicles), cell function (pH of culture medium), and expression of genes related to inflammation (Tnfα) and autophagy (Lc3b). There was no transcriptional change in the anti-apoptotic gene Mcl1, nor the pro-apoptotic gene Hrk, nor in P/GI-unexposed control cultures. LPS-stimulated cells incubated near P/GI had lower expression of the cytokine Il6. These effects were seen as early as 4 h of exposure and at a distance of up to 3 well units from the P/GI-exposed well.ConclusionsExposure to trace residual quantities of P/GI in covered tissue culture plates leads to substantial disruption of cell morphology and function in as little as 4 h, possibly through induction of autophagy but not apoptosis. This phenomenon should be considered when planning time-course experiments in multi-well covered tissue culture plates.

Visual Detection of Chicken Adulteration Based on a Lateral Flow Strip-PCR Strategy.

The aim of this study was to develop an accurate, easy-to-use, and cost-effective method for the detection of chicken adulteration based on polymerase chain reaction (PCR) and lateral flow strip (LFS). We compared six DNA extraction methods, namely the cetyltrimethylammonium bromide (CTAB) method, salt method, urea method, SDS method, guanidine isothiocyanate method, and commercial kit method. The chicken cytb gene was used as a target to design specific primers. The specificity and sensitivity of the PCR-LFS system were tested using a self-assembled lateral flow measurement sensor. The results showed that the DNA concentration obtained by salt methods is up to 533 ± 84 ng µL−1, is a suitable replacement for commercial kits. The PCR-LFS method exhibits high specificity at an annealing temperature of 62 °C and does not cross-react with other animal sources. This strategy is also highly sensitive, being able to detect 0.1% of chicken in artificial adulterated meat. The results of the test strips can be observed with the naked eye within 5 min, and this result is consistent with the electrophoresis result, demonstrating its high accuracy. Moreover, the detection system has already been successfully used to detect chicken in commercial samples. Hence, this PCR-LFS strategy provides a potential tool to verify the authenticity of chicken.

Using discrete event simulation to optimize nucleic acid testing process for coronavirus disease 2019 (COVID-19).

BackgroundThe coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory coronavirus-2 (SARS-CoV-2) has placed enormous diagnostic burden on hospitals and testing laboratories. It is thus critical for such facilities to optimize the diagnostic process to enable maximum testing on minimum resources. The current standard of diagnosis is the detection of the viral nucleic acid in clinical specimens.MethodsIn order to optimize the laboratory’s nucleic acid testing system for COVID-19, we performed a Discrete-Event-Simulation using the Arena Simulation Software to model the detection process based on the data obtained from the First Affiliated Hospital of Guangzhou Medical University (FAHGMU). The maximum of total time that specimens spent and the equipment consumption was compared under different scenarios in the model.ResultsSeven scenarios were performed to simulate actual situation and improved situations. We analyzed conditions that adding a new nucleic acid extraction system (NAES), shifting a member from night duty to morning duty, using specimen tubes containing guanidine isothiocyanate (GITC), then tested the maximum testing capacity in the current number of technicians. In addition, the costs including personal protective equipment (PPE) and testing kits was calculated.ConclusionsA work schedule based on specimen-load improves efficiency without incurring additional costs, while using the specimen tubes containing GITC could reduce testing time by 30 min. In contrast, adding new NAESs or polymerase chain reaction (PCR) instruments has minimal impact on testing efficiency.

A Maximum Flow-Based Approach to Prioritize Drugs for Drug Repurposing of Chronic Diseases.

The discovery of new drugs is required in the time of global aging and increasing populations. Traditional drug development strategies are expensive, time-consuming, and have high risks. Thus, drug repurposing, which treats new/other diseases using existing drugs, has become a very admired tactic. It can also be referred to as the re-investigation of the existing drugs that failed to indicate the usefulness for the new diseases. Previously published literature used maximum flow approaches to identify new drug targets for drug-resistant infectious diseases but not for drug repurposing. Therefore, we are proposing a maximum flow-based protein–protein interactions (PPIs) network analysis approach to identify new drug targets (proteins) from the targets of the FDA (Food and Drug Administration) drugs and their associated drugs for chronic diseases (such as breast cancer, inflammatory bowel disease (IBD), and chronic obstructive pulmonary disease (COPD)) treatment. Experimental results showed that we have successfully turned the drug repurposing into a maximum flow problem. Our top candidates of drug repurposing, Guanidine, Dasatinib, and Phenethyl Isothiocyanate for breast cancer, IBD, and COPD were experimentally validated by other independent research as the potential candidate drugs for these diseases, respectively. This shows the usefulness of the proposed maximum flow approach for drug repurposing.

Evaluation of the SARS-CoV-2 Inactivation Efficacy Associated With Buffers From Three Kits Used on High-Throughput RNA Extraction Platforms.

Rapid and demonstrable inactivation of SARS-CoV-2 is crucial to ensure operator safety during high-throughput testing of clinical samples. The inactivation efficacy of SARS-CoV-2 was evaluated using commercially available lysis buffers from three viral RNA extraction kits used on two high-throughput (96-well) RNA extraction platforms (Qiagen QIAcube HT and the Thermo Fisher KingFisher Flex) in combination with thermal treatment. Buffer volumes and sample ratios were chosen for their optimised suitability for RNA extraction rather than inactivation efficacy and tested against a representative sample type: SARS-CoV-2 spiked into viral transport medium (VTM). A lysis buffer mix from the MagMAX Pathogen RNA/DNA kit (Thermo Fisher), used on the KingFisher Flex, which included guanidinium isothiocyanate (GITC), a detergent, and isopropanol, demonstrated a minimum inactivation efficacy of 1 × 105 tissue culture infectious dose (TCID)50/ml. Alternative lysis buffer mixes from the MagMAX Viral/Pathogen Nucleic Acid kit (Thermo Fisher) also used on the KingFisher Flex and from the QIAamp 96 Virus QIAcube HT Kit (Qiagen) used on the QIAcube HT (both of which contained GITC and a detergent) reduced titres by 1 × 104 TCID50/ml but did not completely inactivate the virus. Heat treatment alone (15 min, 68°C) did not completely inactivate the virus, demonstrating a reduction of 1 × 103 TCID50/ml. When inactivation methods included both heat treatment and addition of lysis buffer, all methods were shown to completely inactivate SARS-CoV-2 inactivation against the viral titres tested. Results are discussed in the context of the operation of a high-throughput diagnostic laboratory.

Comparative study of different SARS-CoV-2 diagnostic techniques

The rapid spread of SARS-CoV-2 led to the necessity of developing diagnostic tests for rapid virus detection. Many commercial platforms have appeared and have been approved for this purpose. In this study, 95 positive and 5 negative retrospective samples were analyzed by 4 different commercial RT-qPCR kits (TaqMan 2019nCoV Assay, Allplex™SARS-COV-2 Assay, FTD SARS-COV-2 Assay and qCOVID-19). The Hologic Aptima SARS-COV-2 and the Clart-COVID-19 system were also tested. serial dilutions of SARS-COV-2 standard control were included for sensitivity analysis. Among the qPCR tested qCOVID19 and Allplex™SARS-COV-2 Assay were both able to detect all the clinical samples included in the study. All four qPCR evaluated showed high sensitivity for samples with Ct<33. Clart-COVID-19 microarrays detected all samples and controls used in this study whereas Hologic Aptima Panther failed with one of the clinical samples. However, the main problem with this system was the number of invalidated samples despite avoiding the use of medium with guanidine isothiocyanate as recommended by the manufacturer. All the techniques tested were of value for SARS-CoV-2 detection.

Rare-Earth Doping Graphitic Carbon Nitride Endows Distinctive Multiple Emissions with Large Stokes Shifts

Rare-Earth Doping Graphitic Carbon Nitride Endows Distinctive Multiple Emissions with Large Stokes Shifts

Sample Exchange to Standardize Molecular Monitoring for CML: The EUTOS Experience

International management recommendations for chronic myeloid leukemia (CML) patients undergoing tyrosine kinase inhibitor therapy emphasise the importance of frequent molecular monitoring to determine the degree of response. Achievement or non-achievement of time-dependent molecular milestones helps to identify individuals who may or may not benefit from a change in treatment. Sequential monitoring enables the timely detection of relapse and may help to highlight other issues influencing response such as compliance.Molecular monitoring is performed by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) for BCR-ABL1 mRNA, and the use of a reference gene helps to control for variation in quality from sample to sample. Guidelines specify that results should be expressed on the International Scale (IS) for BCR-ABL1 measurement, which is essentially the same scale used in the International Randomized Study of Interferon and STI571 (IRIS) study. The ‘gold standard’ method for testing laboratories to generate results on the IS is to apply a laboratory-specific conversion factor (CF) to their local results, with the CF derived by patient sample exchange with an established reference laboratory (Branford et al., Blood 2008). We report here the experience of the European Treatment and Outcome Study (EUTOS) to standardise molecular monitoring in Europe by an expanded programme of sample exchange.The laboratory in Mannheim initially established itself as a European reference laboratory following derivation and validation of their own CF by patient sample exchange with the reference laboratory in Adelaide. Samples exchanges between Mannheim and Adelaide were repeated on a further 5 occasions and confirmed stability of the Mannheim CF. The Mannheim laboratory then undertook further patient sample exchanges with testing laboratories in Europe. Patient samples, typically up to 30 per round, were lysed in Trizol or guanidinium isothiocyanate solution and were provided by both the reference laboratory and the test laboratory. Samples were selected to span the range of detectable disease from approximately 10% BCR-ABLIS to MR4 and were transported on dry ice. Samples were extracted and analyzed in both centres, and results compared. Any consistent bias in the results between the reference laboratory IS values and the test laboratory values was identified by Bland-Altman analysis and a CF defined as the antilog of the mean bias. Multiple rounds of sample exchange were performed to confirm the validity of individual CFs and to help identify changes in test laboratory assay performance over time.The study was performed over a 10 year period (2006-2016) with the participation of 58 laboratories (defined as EUTOS reference centres) from 22 countries. A total of 291 sample exchanges were performed (median = 5; range 1-8), comprising 8396 samples of which 6956 were evaluable (mean 24 successful samples/exchange). Of the 291 sample exchanges, 7 were considered unsatisfactory due to poor concordance (<50% of samples within 2 fold), resulting in the issuance of 284 CF certificates. Of these, 244 (86%) were for laboratories using ABL1 as a reference gene and 44 for those using GUSB. For ABL1 laboratories the median CF was 0.56 (range 0.097-2.87) and for those using GUSB the median was 0.96 (0.32-3.82). Despite the wide range of CF values, 81% and 73% were within 2-fold of the median for ABL1 and GUSB, respectively, and the interquartile ranges were 0.37-0.81 for ABL1 and 0.68-1.34 for GUSB. Using previously described criteria for concordance (Branford et al., Blood 2008; Cross et al. Leukemia 2016), we defined a stable CF as CFn/CFn+1 = 0.63-1.58, with an optimal ratio of 0.83-1.2. Using these criteria, 124 CFs were stable on sequential analysis and 72 were unstable.Overall our study demonstrates the feasibility of large scale sample exchanges to promulgate laboratory-specific CFs to the IS, but the process is onerous, challenging and expensive. We anticipate that sample exchange will remain an option for laboratories to calibrate their assays but is likely to be largely superseded by approaches that use IS-calibrated secondary reference reagents. DisclosuresMüller:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Institute for Hematology and Oncology (IHO GmbH): Employment, Equity Ownership. Dietz:Institute for Hematology and Oncology (IHO GmbH): Equity Ownership. Saglio:Incyte: Consultancy, Honoraria; Roche: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Mahon:NOVARTIS PHARMA: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria; PFIZER: Consultancy, Honoraria; INCYTE: Honoraria. Pane:Novartis: Honoraria, Speakers Bureau. Hochhaus:Incyte: Research Funding; Novartis: Research Funding; MSD: Research Funding; BMS: Research Funding; ARIAD: Research Funding; Pfizer: Research Funding. Cross:Incyte: Consultancy, Honoraria; Qiagen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Ariad: Consultancy, Honoraria.

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is influenced by the type of transport medium: Implications for diagnosis and monitoring.

It is unclear if the use of a molecular transport medium (MTM) containing guanidine isothiocyanate (GITC) would be advantageous over the CDC recommended, commonly used viral transport medium (VTM). We retested 70 SARS‐CoV2 cases by RT‐PCR in varying stages of follow‐up using MTM and VTM in parallel and found discrepant results of RNase P, E and N genes. Majority (81%) patients tested positive with MTM as compared with VTM (27.1%). Even patients who were sampled 3 weeks after diagnosis demonstrated a significant discrepancy in the positivity rates between MTM vs VTM raising concerns about the clinical utility of VTM.

A simple, safe and sensitive method for SARS-CoV-2 inactivation and RNA extraction for RT-qPCR.

The SARS‐CoV‐2 pandemic has created an urgent need for diagnostic tests to detect viral RNA. Commercial RNA extraction kits are often expensive, in limited supply, and do not always fully inactivate the virus. Together, this calls for the development of safer methods for SARS‐CoV‐2 extraction that utilize readily available reagents and equipment present in most standard laboratories. We optimized and simplified a RNA extraction method combining a high molar acidic guanidinium isothiocyanate (GITC) solution, phenol and chloroform. First, we determined the GITC/RNA dilution thresholds compatible with an efficient two‐step RT‐qPCR for B2M mRNA in nasopharyngeal (NP) or oropharyngeal (OP) swab samples. Second, we optimized a one‐step RT‐qPCR against SARS‐CoV‐2 using NP and OP samples. We furthermore tested a SARS‐CoV‐2 dilution series to determine the detection threshold. The method enables downstream detection of SARS‐CoV‐2 by RT‐qPCR with high sensitivity (~4 viral RNA copies per RT‐qPCR). The protocol is simple, safe, and expands analysis capacity as the inactivated samples can be used in RT‐qPCR detection tests at laboratories not otherwise classified for viral work. The method takes about 30 min from swab to PCR‐ready viral RNA and circumvents the need for commercial RNA purification kits.

Genetic Identification of Edible Bird's Nest in Thailand Based on ARMS-PCR.

Edible bird’s nest (EBN) is a popular delicacy in the Asian Pacific region originating from Indonesia, Malaysia, Thailand and Vietnam, which consist of various potential medicine value in Traditional Chinese Medicine (TCM). Thailand is one of the main exporters of EBN. However, the genetic information of EBN, a key part of molecular biology, has yet to be reported in Thailand. It is necessary to explore the genetic information of EBN in Thailand based on a quick and simple method to help protect the rights and interests of consumers. This research aimed to systematically evaluate different methods of extracting EBN DNA to improve the efficiency of the analysis of cytochrome b (Cytb) and NADH dehydrogenase subunit 2 (ND2) gene sequences, the establishment of phylogenetic trees, and the genetic information of EBN in Thailand. Additionally, we aimed to develop a quick and simple method for identifying EBN from different species based on the genetic information and amplification-refractory mutation system PCR (ARMS-PCR). By comparing the four methods [cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), kit and guanidinium isothiocyanate methods] for EBN extraction, we found that the guanidinium isothiocyanate method was the optimal extraction method. Phylogenetic trees generated on the basis of Cytb and ND2 gene analyses showed that 26 samples of house EBN and 4 samples of cave EBN came from Aerodramus fuciphagus and Aerodramus maximus, respectively. In addition, to distinguish different samples from different species of Apodiformes, we designed 4 polymerase chain reaction (PCR) amplification primers based on the ND2 gene sequences of A. fuciphagus and A. maximus. The ARMS-PCR results showed band lengths for A. fuciphagus EBN of 533, 402, and 201 bp, while those for A. maximus EBN were 463, 317, and 201 bp. Collectively, the results showed that ARMS-PCR is a fast and simple method for the genetic identification of EBN based on designing specific original identification primers.

RNA Isolation from Articular Cartilage Tissue.

Isolation of high-quality RNA directly from tissues is desirable to obtain precise information of in vivo gene expression profiles in cells embedded within their extracellular matrix (ECM). It is well known that purification of RNA from cartilage tissues is particularly challenging due to low cell (chondrocyte) content and its dense ECM rich in large negatively charged proteoglycans that can copurify with RNA. Older methodologies to purify RNA from cartilage involved the use of concentrated denaturing solutions containing guanidinium isothiocyanate followed by ultracentrifugation in cesium trifluoroacetate. Such ultracentrifugation approaches are rarely used now since the emergence of more user-friendly mini spin column chromatography kits. For this chapter, we tested and compared three methods to isolate RNA from immature murine articular (femoral head) cartilage and found that the combination of TRIzol® reagent and spin column chromatography (Norgen Total RNA Purification Kit) was the best approach to generate higher quality RNA. Here, the average RNA Integrity Number (RIN), as determined by Bioanalyzer technology, was 7.1. We then applied this method to attempt to isolate RNA directly from human articular cartilage harvested from three osteoarthritic (OA) knee joint specimens. As expected, the concentration and quality of RNA obtained differed between samples. However, from one specimen, we were able to isolate approximately 3μg of total RNA (including small noncoding RNAs) from 100mg of human OA cartilage with a RIN=7.9. Despite the patient-to-patient variabilities that are known to exist between cartilage specimens from OA joints, we have demonstrated that it is possible to obtain reasonably high levels of RNA from human OA articular cartilage at a quality suitable for downstream analyses including microarray and RNA-Seq. A detailed description of our preferred RNA purification methodology, which can be used to isolate RNA from human, bovine, or rodent cartilage tissue, is provided in this chapter.

Demonstration of a fast and easy sample-to-answer protocol for tuberculosis screening in point-of-care settings: A proof of concept study.

We sought to develop a smooth and low cost sample preparation and DNA extraction protocol, streamlined with a ready-to-use qPCR in a portable instrument to overcome some of the existing hurdles. Several solutions were evaluated as to their ability to liquefy a mucin-based matrix. Each liquefied matrix, supplemented with either Mycobacterium tuberculosis (MTB) H37Rv strain DNA or intact cells, was aliquoted onto a filter paper embedded with solubilizing agents, and was subsequently dried up. Most of the nucleic acids, including genomic DNA from the bacilli and the host, binds to the filter paper. Next, several protocols were evaluated to elute the DNA from the paper, using qPCR to detect the insertion sequence IS6110, a M. tuberculosis complex genomic marker. The limit of detection (LOD) of the best protocol was then evaluated using parallel seeding and colony counting. The protocol was also evaluated using seventeen sputum samples, previously characterized by the GeneXpert or culture. Two instruments (the ABI7500 Standard and the Q3-Plus system) and two reagents storage formats (frozen or ready-to-use) were evaluated. Solutions containing guanidine isothiocyanate exerted the best liquefying effect on the mucin-based matrix extracted from one 6-mm punches, followed by a brief incubation at 95°C. The resulting DNA contained impurities, but a simple 1:10 dilution elicited the detection of MTB and human genomic targets. The described protocol presented an apparent LOD of 02 CFU/mL of MTB. Challenging the protocol with previously characterized samples showed substantial agreement with GeneXpert MTB/RIF results (sensitivity of 90%, agreement of 88.9%, kappa coefficient of 0.77), and moderate agreement with culture results (sensitivity of 100%, agreement of 78.9%, kappa coefficient of 0.58). This work presents a sensitive proof-of-concept protocol for sputum liquefaction and decontamination followed by a simple DNA extraction procedure, in which the extraction steps are streamlined with a ready-to-use qPCR in a portable instrument that can be employed in low infrastructure settings.

DNA Extraction with TRIzol Reagent Using a Silica Column.

TRIzol is a monophasic solution of phenol and guanidine isothiocyanate used for the extraction of RNA, DNA and proteins from tissues or cells. However, few studies have described its application to DNA extraction due to its time-consuming procedure. We present a TRIzol-modified method of extracting DNA from tissues using the TRIzol reagent and a silica column, which requires only one-third of the time required for the classic extraction procedure. Spectrophotometric analysis showed that the 260/280 and 260/230 nm optical density ratio of the DNA extracted using the TRIzol-modified method is ideal and equal to that obtained by the classic method and commercial DNAiso methods. The DNA extracted by the TRIzol-modified method had the same performance in a restriction enzyme digestion and quantitative PCR as that extracted using the classic method. Using the TRIzol-modified method saves time, simplifies the DNA extraction procedure, and facilitates various molecular biology assays.