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Colistin HiMIC Plate Kit (HiMedia Laboratories), a new commercial broth microdilution (BMD) test for colistin susceptibility testing was evaluated. BMD according to ISO standard 20776–1 (2019) with two-fold dilutions from 128 to 0.125 mg/L was used as a reference method. The colistin reference MICs (minimal inhibitory concentration) ranged from 0,25 to 128 mg/L with 15 (20.5%; 15/73) isolates having colistin reference MICs close to the current EUCAST breakpoint (MICs of 2, 4, and 8 mg/L). The study assessed the compliance of a commercial kit with the CLSI criteria, including categorical agreement (CA) and essential agreement (EA ≥90%), very major error (VME rate) <3%, and major error (ME) rate <3%. On 73 carbapenemase-producing Klebsiella pneumoniae isolates Colistin HiMICTM Plate Kit showed CA and EA of 100% (73/73; 95% CI: 0.97–1.00) and 82.2% (60/73; 95% CI: 0.72–0.90), respectively. No ME (false-resistant results) and VME (false-susceptible results) were detected. Kit showed acceptable CA, ME, and VME error parameters, whereas the EA did not meet the ≥90% threshold. Laboratories must check for possible limitations of commercial kits before they can be used for colistin susceptibility testing. [ABSTRACT FROM AUTHOR]

Objective: Measurements utilizing commercially available sets of reagents for determination of steroid hormone profiles by liquid chromatography–tandem mass spectrometry (LC-MS/MS) have become increasingly important for routine laboratories. However, method-specific publications of reference intervals obtained from sufficiently large studies are often missing. Methods: After validation of performance characteristics, a widely available kit for steroid analysis by LC-MS/MS was used to measure concentrations of 15 endogenous steroids (aldosterone, cortisol, cortisone, corticosterone, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone sulfate, estradiol, testosterone, androstenedione, dihydrotestosterone, dehydroepiandrosterone, 17-hydroxyprogesterone, 11-deoxycorticosterone, progesterone) in more than 500 blood samples from a population-based study. While randomly selected from a larger cohort, the samples equally represented both sexes and covered a wide range of adult age groups. Age- and sex-specific reference intervals were calculated, and correlation with BMI was assessed. Results: Performance characteristics of the assay matched expectations for 9 of 15 steroids. For most of them, reference intervals obtained from our study population were comparable to those reported by others, with age and sex being the major determinants. A sex-specific correlation with BMI was found for seven steroids. We identified limitations regarding sensitivity of the method for quantification of progesterone in males and postmenopausal females. Concentrations of aldosterone, 21-deoxycortisol, estradiol, 11-deoxycorticosterone, and dihydrotestosterone could not be quantified in a large percentage of samples. Conclusions: The reference intervals for nine steroids will support meaningful interpretation for steroid profiles as measured by a widely used kit for LC-MS/MS-based quantification. Laboratories using such kits must be aware of potential limitations in sensitivity for some steroids included in the profile. Significance Statement Quantification of steroid hormones is a cornerstone for diagnosis of several diseases. Commonly used immunoassays have limitations in specificity. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) is a promising alternative, particularly if methods are harmonized across laboratories. The use of kits from commercial suppliers might support this. Clinical interpretation of steroid concentrations requires availability of appropriate reference intervals (RIs), but studies on RIs reported in the literature differ in preanalytical and analytical procedures. Here, we provide RIs for steroids measured by a widely available kit under preanalytical conditions mirroring common clinical practice. Such RIs might facilitate interpretation for those using the same method and comparable conditions in clinical routine.

Abstract RNA extraction from Mycobacterium tuberculosis has been a historically challenging task for researchers due to the thick lipids associated with the cell wall of this “notorious” pathogen that is responsible for Tuberculosis (TB) outbreaks. Several studies have successfully extracted RNA from M. tuberculosis using a Trizol reagent combined with organic solvents. Recently, our laboratory has successfully extracted high quality total RNA using a commercial kit from clinical strains belonging to F15/LAM4/KZN, Beijing and F11 strain families and H37Rv laboratory strain by exploiting high speed homogenizer for cell lysis and spin columns for RNA purification. The quality and integrity of the extracted RNA was analyzed and confirmed through the Nanodrop, Bioanalyzer and RNA 3-(N-morpholino) propanesulfonic acid (MOPS) gel electrophoresis. Furthermore, to confirm the integrity of small RNA (sRNA) molecules due to their vulnerability to degradation, the RNA samples were converted to cDNA and sRNAs were amplified and confirmed through PCR. This detailed RNA extraction protocol proposes to carve a new path into TB transcriptome research without the use of organic solvent for downstream purification steps while yielding high quality RNA that can be used to understand M. tuberculosis transcriptome regulation.

RNA extraction from Mycobacterium tuberculosis has been a historically challenging task for researchers due to the thick lipids associated with the cell wall of this "notorious" pathogen that is responsible for Tuberculosis (TB) outbreaks. Several studies have successfully extracted RNA from M. tuberculosis using a Trizol reagent combined with organic solvents. Recently, our laboratory has successfully extracted high quality total RNA using a commercial kit from clinical strains belonging to F15/LAM4/KZN, Beijing and F11 strain families and H37Rv laboratory strain by exploiting high speed homogenizer for cell lysis and spin columns for RNA purification. The quality and integrity of the extracted RNA was analyzed and confirmed through the Nanodrop, Bioanalyzer and RNA 3-(N-morpholino) propanesulfonic acid (MOPS) gel electrophoresis. Furthermore, to confirm the integrity of small RNA (sRNA) molecules due to their vulnerability to degradation, the RNA samples were converted to cDNA and sRNAs were amplified and confirmed through PCR. This detailed RNA extraction protocol proposes to carve a new path into TB transcriptome research without the use of organic solvent for downstream purification steps while yielding high quality RNA that can be used to understand M. tuberculosis transcriptome regulation. [ABSTRACT FROM AUTHOR]

Introduction: The market offers a wide range of extracellular vesicles (EVs) isolation products, but their lack of standardization is a concern. Therefore, it is important to carefully assess the quality of the EVs obtained using these products. In this study, we compared the EXOCIB kit with the ultracentrifuge method, which is considered the gold standard for small EV isolation., Methods: After overnight fasting, small plasma EVs were extracted from four individuals using both the ultracentrifuge and the EXOCIB kit methods. The pooled EVs were then compared for the presence of the cluster of differentiation 63 (CD63) protein using the western blot analysis, and their size and zeta potential were performed by Dynamic Light Scattering (DLS). In addition, the size and morphology of small EVs were determined by using the Transmission Electron Microscopy (TEM) technique., Results: An average hydrodynamic size of 135.7 nm and a zeta potential of -6.33 Mv at 25°C was found for small EVs isolated by the ultracentrifuge, whereas the kit method resulted in small EVs with a hydrodynamic size of 102.8 nm and a zeta potential of -0.907. Notably, the size of the particles in the kit samples was smaller compared to those obtained through the ultracentrifuge (P < 0.001). The western blot method confirmed the expression of CD63 in both methods, so the ultracentrifuge yielded small EVs with a higher level of purity compared to the kit-based approach (P = 0.036)., Conclusion: The DLS findings revealed the existence of vesicles within the appropriate size range for small EVs like exosomes in both isolation techniques. The results of the western blot analysis, in conjunction with DLS, displayed that the ultracentrifuge method extracted small EVs with a greater degree of purity than the kit-based approach., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Exosomes are extracellular endosomal nanoparticles, which are formed under complex processes during the formation of multivesicular bodies. They are also achieved from conditioned media of a variety of cell types, especially mesenchymal stem cells (MSCs). Exosomes can modulate intracellular physiological actions via signaling molecules on the surface or secretion of components to the extracellular spaces. Furthermore, they are potentially used as crucial agents for cell-free therapy; however, their isolation and characterization can be challenging. In the current study, two methods of exosome isolation have been characterized and compared using a culture media of adipose-derived mesenchymal stem cells, namely ultracentrifugation and a commercial kit; moreover, the efficiency of these two methods was highlighted in this study. Two different isolation methods of exosomes from MSCs were used to compare the efficiency of exosomes. For both isolation methods, transmission electron microscopy, dynamic light scattering (DLS), and bicinchoninic acid (BCA) assay have been performed. The electron microscopy and DLS indicated the presence of exosomes. Moreover, the kit and ultracentrifugation isolates contained approximately comparable amounts of protein measured by the BCA. Overall, the two isolation methods had similar performances. Although ultracentrifugation is used as a gold standard for exosome isolation, the commercial kit has some advantages and can be applied alternatively according to its cost-effectiveness and time-saving properties. [ABSTRACT FROM AUTHOR]

Bovine leukemia virus (BLV) is an oncogenic virus belonging to the genus Deltaretrovirus and is the causative agent of enzootic bovine leukosis. Proviral load (PVL) determined by real-time quantitative PCR (qPCR) is now widely used as an indicator of not only BLV infection, but also BLV disease progression. To interpret PVLs determined by different qPCRs used in Japan, we compared a chimeric cycling probe-based qPCR, CY415, targeting the BLV tax region; a TaqMan probe-based qPCR, RC202, targeting the BLV pol region; and a TaqMan probe-based qPCR, CoCoMo, targeting the BLV long terminal repeat (LTR) region. Whole-blood samples collected from 317 naturally BLV-infected cattle (165 Holstein–Friesian and 152 Japanese Black) and tumor tissue samples collected from 32 cattle at a meat inspection center were used. The PVLs determined by each qPCR were strongly correlated. However, the PVL and the proportion of BLV-infected cells determined by RC202 or CoCoMo were significantly higher than those determined by CY415. Genetic analysis of three tumor tissue samples revealed that LTR region mutations or a deletion affected the PVL determined by CoCoMo. These results suggest that the TaqMan-based RC202 or CoCoMo qPCR is better than CY415 for BLV PVL analysis. However, qPCR target region mutations were not rare in tumors and could hamper PVL analysis by using qPCR. [ABSTRACT FROM AUTHOR]

This study was performed to compare the sensitivity of seven plate methods and commercial kits for the detection of antibiotics in vitro and in poultry carcasses. In the first step, different concentrations of five commonly used antibiotics in poultry breeding were prepared and the sensitivity of both methods in antibiotic residue detection was evaluated. Subsequently, 20 days old broilers were divided into 5 groups. After an adaptation period and feeding non-antibiotic diet, groups were treated with therapeutic doses of Oxytetracycline, Enrofloxacin, Florfenicol, and Fosfomycin through drinking water. One group was considered as a control. At antibiotic discontinuation day (day 0) and days 1, 2, 3, 4, 5, and 6 thereafter, 3 chicks from each group were slaughtered and thigh muscle, liver and kidney were sampled and residual antibiotics were evaluated by both methods. The results showed that sensitivity of commercial kits was higher in in-vitro condition than that of poultry carcass. In the seven plate method, the residuals were more detectable in the liver and kidney. The commercial kit was able to detect antibiotics only up to 24 hours, after which almost all specimens were negative. The seven plate method was positive for most of the specimens up to three days after discontinuation. This study showed that the efficacy of the seven-plate method was more than commercial kits, and in order to increase the efficiency of detection methods, it is recommended to perform it on the liver and kidney of four carcasses from each herd (total of 8 samples). If at least four samples are positive, the herd is considered as infected and the HPLC method is used to confirm the specimens. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Faculty of Pharmacy of Ankara University / Ankara Üniversitesi Eczacilik Fakültesi Dergisi is the property of Ankara University and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

BACKGROUND: Antibiotics are widely used for the treatment of livestock. Their inappropriate usage leads to various disorders in humans as a result of consuming animal products. Milk is among the foods that are significantly affected by consuming antibiotics. OBJECTIVES: The objective of this study was to compare Yoghurt Culture Test (YCT), Four- Plate Test (FPT), and the Copan test for detecting antibiotic residues in raw and pasteurized milk produced in Chaharmahal and Bakhtiari province, Iran. METHODS: A total of 146 raw milk samples and 54 pasteurized milk samples were selected randomly from dairy farms and dairy products suppliers. The presence of antibiotics was evaluated by YCT, FPT, and Copan test. In addition, the sensi-tivity of the three tests for tetracycline and penicillin, as the two common antibiotics in the treatment of animals, was compared. RESULTS: Our findings showed that 8.9% of raw milk and 11% of pasteurized milk samples contained antibiotics. However, the levels of antibiotic residues were higher in 2% of the positive samples than maximum residue levels (MRL). Moreover, significant differences were observed between FPT, YCT, and the Copan test (p 0.05). CONCLUSIONS: The results of this study showed that a low percentage of milk samples contained antibiotic residues higher than the permissible limit. Furthermore, YCT could be used as an inexpensive, easy, and sensitive method for identifying the residues of penicillin and tetracycline in milk.

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Rotavirus is the important etiological agents of infectious diarrhea among children under 5 years old. Rotaviruses are divided into 10 serogroups (A–J) and each group is based on genetic properties of major structural protein VP6. We designed a novel VP6 sequence optimization to increase the expression level of this protein. Numerous factors such as codon adaptation index, codon pair bias, and guanine‐cytosine content were adapted based on Escherichiacoli codon usage. In addition, the ribosome binding site (RBS) of pET‐15b was redesigned by the RBS calculator and the secondary structure of VP6 messenger RNA was optimized in the whole length of the coding sequence. Various factors including isopropyl beta‐ d‐thiogalactoside (IPTG) concentration, temperature, and induction time were analyzed for the optimization of the best expression in E. coli by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and western blotting. The recombinant VP6 (rVP6) protein was purified by the Ni‐sepharose and then the hyperimmune sera were generated against rVP6 in rabbits. Among three different temperatures, IPTG concentrations, and postinductions, the level of rVP6 was higher at 37°C, 1 mM of IPTG, and 8 h, respectively. Also, the high expression level of rVP6 was obtained in the insoluble aggregate form (43.8 g/L). After purification, the yield of rVP6 was 10.83 g/L. The rVP6 specific antiserum was confirmed by both immunofluorescent and western blotting. The versatile sequence optimization was the reason to produce a high level of rVP6 compared to other reports and can potentially apply to produce cheaper commercial kits to diagnose serological tests and new rotavirus vaccines. Highlights: The versatile sequence optimization was the reason to produce a high level of rVP6.The high yield of rVP6 can apply to produce cheaper commercial kit.Polyclonal antibody was raised against rVP6.Immunoblotting and immunofluorescence assays showed the potency of VP6 antibody to detect RV. [ABSTRACT FROM AUTHOR]

• Species specificity of commercial human DNA assay kits was examined. • Cross-species amplifications of non-human primate DNAs is observed. • Species specificity differed among the examined kits. • Cross-species data will aid human identification in forensic cases involving non-human primates. Species specificity of commercial human DNA quantification kits and short tandem repeat (STR) profiling kits was examined using primate DNA samples. These samples comprised 33 individuals from eight primate species, each with gender and kinship data, including human (Homo sapiens), chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus) of Hominidae family, and Japanese macaque (Macaca fuscata), long-tailed macaque (Macaca fascicularis), hamadryas baboon (Papio hamadryas), and savannah monkey (Chlorocebus sp.) of Cercopithecidae family. The findings revealed varying levels of cross-species amplifications in all non-human DNA samples that correlated with their evolutionary proximity to humans, both kit types. Moreover, cross-species amplification, including female DNA samples, was observed in a Y-chromosomal STR profiling kit. Additionally, species specificity differed among the commercial kits examined. The cross-species amplification data presented in this study offer valuable assistance in interpreting the results of individual human identification in forensic cases involving non-human primates. [ABSTRACT FROM AUTHOR]

Bovine leukemia virus (BLV) is an oncogenic virus belonging to the genus Deltaretrovirus and is the causative agent of enzootic bovine leukosis. Proviral load (PVL) determined by real-time quantitative PCR (qPCR) is now widely used as an indicator of not only BLV infection, but also BLV disease progression. To interpret PVLs determined by different qPCRs used in Japan, we compared a chimeric cycling probe-based qPCR, CY415, targeting the BLV tax region; a TaqMan probe-based qPCR, RC202, targeting the BLV pol region; and a TaqMan probe-based qPCR, CoCoMo, targeting the BLV long terminal repeat (LTR) region. Whole-blood samples collected from 317 naturally BLV-infected cattle (165 Holstein–Friesian and 152 Japanese Black) and tumor tissue samples collected from 32 cattle at a meat inspection center were used. The PVLs determined by each qPCR were strongly correlated. However, the PVL and the proportion of BLV-infected cells determined by RC202 or CoCoMo were significantly higher than those determined by CY415. Genetic analysis of three tumor tissue samples revealed that LTR region mutations or a deletion affected the PVL determined by CoCoMo. These results suggest that the TaqMan-based RC202 or CoCoMo qPCR is better than CY415 for BLV PVL analysis. However, qPCR target region mutations were not rare in tumors and could hamper PVL analysis by using qPCR.

BACKGROUND: Antibiotics are widely used for the treatment of livestock. Their inappropriate usage leads to various disorders in humans as a result of consuming animal products. Milk is among the foods that are significantly affected by consuming antibiotics. OBJECTIVES: The objective of this study was to compare Yoghurt Culture Test (YCT), Four- Plate Test (FPT), and the Copan test for detecting antibiotic residues in raw and pasteurized milk produced in Chaharmahal and Bakhtiari province, Iran. METHODS: A total of 146 raw milk samples and 54 pasteurized milk samples were selected randomly from dairy farms and dairy products suppliers. The presence of antibiotics was evaluated by YCT, FPT, and Copan test. In addition, the sensitivity of the three tests for tetracycline and penicillin, as the two common antibiotics in the treatment of animals, was compared. RESULTS: Our findings showed that 8.9% of raw milk and 11% of pasteurized milk samples contained antibiotics. However, the levels of antibiotic residues were higher in 2% of the positive samples than maximum residue levels (MRL). Moreover, significant differences were observed between FPT, YCT, and the Copan test (P<0.05). On the other hand, the positive results of YCT and Copan tests were not significantly different (P>0.05). CONCLUSIONS: The results of this study showed that a low percentage of milk samples contained antibiotic residues higher than the permissible limit. Furthermore, YCT could be used as an inexpensive, easy, and sensitive method for identifying the residues of penicillin and tetracycline in milk. [ABSTRACT FROM AUTHOR]

The genomes of most known mycoviruses consist of double stranded RNA (dsRNA) or single stranded RNA (ssRNA). Therefore, for all aspects of mycovirology, the research is highly dependent on the quality and quantity of RNA either by the extraction of genomic dsRNA or dsRNA as a replicating intermediate. A common procedure to extract dsRNA is its binding on a cellulose matrix after a phenol/chloroform purification step. A commercial kit for dsRNA extraction facilitated the researchers´ daily work, but is not available anymore. To extract nucleic acids in a standardized good quality and quantity from small amounts of starting material, we compared commercial kits for gDNA extraction to the kits for RNA extraction using fungal material with a high and a low virus titer. Here we show that viral dsRNA can be extracted using commercial gDNA kits from fungal tissue with a high and a low virus titer in the same quality and quantity as it was done with the discontinued dsRNA extraction kit. • Viral dsRNA is extracted by commercial gDNA kits. • Viral dsRNA is comparable to samples extracted by a dsRNA kit. • Low titer infections can be detected by agarose gel electrophoresis. [ABSTRACT FROM AUTHOR]

The most suitable method of total DNA extraction still remains the crucial step for many plant species, although there are many different protocols and commercial kits for DNA isolation. In this study, five different extraction protocols were analysed to find out the most appropriate method for DNA extraction from Hedera helix L. This species has numerous medical and pharmaceutical uses and is also characterized by antioxidant effects on human body. In spite of its wide medical utilization, it belongs to those plant species, where the genomic information is very limited. Comparing of different protocols resulted in the yield of extracted DNA that has ranged from 6.3 to 487 ng μl-1. The purity of extracted DNA has ranged from 1.4 up to 2.0 A260/A280. All the extraction methods used in this study were evaluated not only in term of quantity and purity of DNA but also its functionality in the restriction endonuclease digestion and polymerase chain reaction based downstream analysis was performed.

Objective: Measurements utilizing commercially available sets of reagents for determination of steroid hormone profiles by liquid chromatography-tandem mass spectrometry (LC-MS/MS) have become increasingly important for routine laboratories. However, method-specific publications of reference intervals obtained from sufficiently large studies are often missing., Methods: After validation of performance characteristics, a widely available kit for steroid analysis by LC-MS/MS was used to measure concentrations of 15 endogenous steroids (aldosterone, cortisol, cortisone, corticosterone, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone sulfate, estradiol, testosterone, androstenedione, dihydrotestosterone, dehydroepiandrosterone, 17-hydroxyprogesterone, 11-deoxycorticosterone, progesterone) in more than 500 blood samples from a population-based study. While randomly selected from a larger cohort, the samples equally represented both sexes and covered a wide range of adult age groups. Age- and sex-specific reference intervals were calculated, and correlation with BMI was assessed., Results: Performance characteristics of the assay matched expectations for 9 of 15 steroids. For most of them, reference intervals obtained from our study population were comparable to those reported by others, with age and sex being the major determinants. A sex-specific correlation with BMI was found for seven steroids. We identified limitations regarding sensitivity of the method for quantification of progesterone in males and postmenopausal females. Concentrations of aldosterone, 21-deoxycortisol, estradiol, 11-deoxycorticosterone, and dihydrotestosterone could not be quantified in a large percentage of samples., Conclusions: The reference intervals for nine steroids will support meaningful interpretation for steroid profiles as measured by a widely used kit for LC-MS/MS-based quantification. Laboratories using such kits must be aware of potential limitations in sensitivity for some steroids included in the profile., Significance Statement: Quantification of steroid hormones is a cornerstone for diagnosis of several diseases. Commonly used immunoassays have limitations in specificity. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a promising alternative, particularly if methods are harmonized across laboratories. The use of kits from commercial suppliers might support this. Clinical interpretation of steroid concentrations requires availability of appropriate reference intervals (RIs), but studies on RIs reported in the literature differ in preanalytical and analytical procedures. Here, we provide RIs for steroids measured by a widely available kit under preanalytical conditions mirroring common clinical practice. Such RIs might facilitate interpretation for those using the same method and comparable conditions in clinical routine.

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Food fraud has been and still is a problem in the food industry. It is detectable by several approaches, such as high performance liquid chromatography (HPLC), chemometric assays, or DNA-based techniques, each with its own drawbacks. This work addresses one major drawback of DNA-based methods, in particular their sensitivity to inhibitors contained in particular matrices from which DNA is isolated. We tested five commercial kits and one in-house method characterized by different ways of sample homogenization and DNA capture and purification. Using these methods, DNA was isolated from 10 different fruit species commonly used in plant-based foodstuffs. The quality of the DNA was evaluated by UV-VIS spectrophotometry. Two types of qPCR assays were used for DNA quality testing: (i) Method specific for plant ITS2 region, (ii) methods specific for individual fruit species. Based mainly on the results of real-time PCR assays, we were able to find two column-based kits and one magnetic carrier-based kit, which consistently provided fruit DNA isolates of sufficient quality for PCR-based assays useful for routine analysis and identification of individual fruit species in food products.

Urinary cell-free DNA (cfDNA) is an attractive body fluid for liquid biopsy. In this study, we compared the efficiencies of four commercial kits for urinary cell-free DNA (cfDNA) isolation and of various sample storage conditions. Urinary cfDNA was isolated from 10 healthy individuals using four commercial kits: QIAamp Circulating Nucleic Acid Kit (QC; Qiagen), MagMAX™ Cell-Free DNA Isolation Kit (MM; Applied Biosystems), Urine Cell-Free Circulating DNA Purification Midi Kit (NU; Norgen Biotek), and Quick-DNA™ Urine Kit (ZQ; Zymo Research). To assess the isolation efficiency, an Agilent 2100 Bioanalyzer with High Sensitivity DNA chips was used, and cfDNA yield was defined as the amount of cfDNA obtained from 1 mL of urine. MM and QC provided the highest cfDNA yield in the 50–300 bp range, and MM and NU gave the highest cfDNA yield in the 50–100 bp range. In particular, the NU kit was efficient for isolation of more fragmented cfDNA in the range of 50–100 bp with the lowest cellular genomic DNA contamination. ZQ had the best cost-efficiency for isolating the same amount of urinary cfDNA. Samples stored at −70 °C with the addition of 10 mM EDTA resulted in the highest cfDNA yield 3 months after sample collection.

For in-depth characterization of the microbiota associated with shrimp larvae, careful selection of DNA isolation procedure is paramount for avoiding biases introduced in community profiling. Four E.Z.N.A.™ DNA extraction kits, i.e., Bacterial, Mollusc, Stool, and Tissue DNA Kits, abbreviated as Ba, Mo, St, and Ti, respectively, were initially evaluated with zoea 2 (Z2) larvae of the Pacific white shrimp (Litopenaeus vannamei) by 16S amplicon sequencing on a Illumina MiSeq platform. Further characterization of additional larval samples, specifically nauplii 5 (N5), mysis 1 (M1), and postlarvae 1 (P1), was performed with Ba and St kits to examine the changing microbiota profile during shrimp hatchery period. The results from the Z2 samples showed that DNA yields from the four kits varied significantly (P < 0.05), whereas no significant differences were detected in the α-diversity metrics of the microbiota. By contrast, the St kit, with the lowest DNA yield and quality, successfully recovered DNA from Gram-positive and gut-associated bacterial groups, whereas the Ba kit, which showed maximal microbiota similarity with the Mo kit, manifested the best reproducibility. Notably, significant differences were observed in relative abundances of most dominant taxa when comparing results from the Ba and St kits on Z2, M1, and P1 samples. In addition, the bacterial community identified shifted markedly with larval development regardless of the DNA extraction kits. The DNA recovery biases arising from the larval microbiota could be due to different protocols for cell lysis and purification. Therefore, combined application of different DNA extraction methods may facilitate identification of some biologically important groups owing to their complementary effects. This approach should receive adequate attention for a thorough understanding of the larvae-associated microbiota of the penaeid shrimp.

The main goal of any DNA extraction procedure is to ensure reliable and reproducible results in a simple, fast and inexpensive manner. When it comes to plant tissues, this goal is challenging to achieve due to the presence of a variety of metabolites that interfere with DNA during isolation and downstream analysis. In this study, we compared the efficiency of three methods for DNA extraction from plum kernels: 1) the standard CTAB Soltis method which is the most common protocol for DNA extraction from various plant tissues (seeds, young leafs, mature leafs, root); 2) CTAB-based method originally described for DNA isolation from medicinal plants with high levels of secondary metabolites; 3) and one of various commercially available kits. The usefulness of the obtained DNA was evaluated by SSR analysis with seven microsatellite markers. Although the latter two extraction protocols retrieved genomic DNA that gave positive PCR results, only DNA isolated by kit produced full SSR profile.

Purpose: To introduce a novel practical technique of self-made cryopreservative fibrin glue (SMC) applied in pterygium surgery and to assess its safety and efficacy.Methods: Forty-eight eyes of 48 patients with nasal primary pterygium were enrolled. The patients were equally assigned to 6 groups. Self-made fibrin glue was subpackaged and, respectively, cryopreserved for 3, 7, 15 days and 1, 2 and 3 months. At each time point, the asepsis of SMC was confirmed by bacterial culture and colony counting. In each group, corresponding SMC was applied to fix the autograft after the pterygium was removed (e.g., SMC 3d for group 1 and SMC 3m for group 6). All the patients were followed up postoperatively on days 1, 3, 7 and 14 and then at months 1, 3, 6. The main outcome measures included fixation success rate within two tries, postoperative discomfort, recurrence rate and complications.Results: No colony growth was observed in all the fibrinogen and thrombin tubes sent. Five patients needed a second try with respective SMC during the autograft fixation, and there were no significant differences in SMC use times among the groups (P = 0.885). There were no significant differences in postoperative discomfort (day 1, 3, 7; P = 0.651, P = 0.269, P = 0.180, respectively) among the groups. By the end of 6-m follow-up, no infections and severe complications were observed in any group. The total recurrence rate was 3/48 (6%), and there were no significant differences in recurrences among the groups (P = 1.000).Conclusion: SMC is safe and effective for autograft fixation in pterygium surgery. This new practical technique will benefit the patients and surgeons in developing and underdeveloped country. [ABSTRACT FROM AUTHOR]

The main goal of any DNA extraction procedure is to ensure reliable and reproducible results in a simple, fast and inexpensive manner. When it comes to plant tissues, this goal is challenging to achieve due to the presence of a variety of metabolites that interfere with DNA during isolation and downstream analysis. In this study, we compared the efficiency of three methods for DNA extraction from plum kernels: 1) the standard CTAB Soltis method which is the most common protocol for DNA extraction from various plant tissues (seeds, young leafs, mature leafs, root); 2) CTAB-based method originally described for DNA isolation from medicinal plants with high levels of secondary metabolites; 3) and one of various commercially available kits. The usefulness of the obtained DNA was evaluated by SSR analysis with seven microsatellite markers. Although the latter two extraction protocols retrieved genomic DNA that gave positive PCR results, only DNA isolated by kit produced full SSR profile. [ABSTRACT FROM AUTHOR]

Toxoplasmosis is generally a benign infection caused by the protozoan parasite Toxoplasma gondii but can have severe consequences in fetuses of mothers infected during pregnancy (congenital toxoplasmosis) and immunocompromised individuals. PCR-based diagnostic tests have become crucial for its diagnosis. However, this molecular diagnosis essentially relies upon laboratory-developed methods and suffers from a lack of standardization, leading to great variation in methods and performance among laboratories. With the need for accreditation of clinical microbiological laboratories, the use of commercial PCR kits has become an attractive alternative; but thorough evaluation of newly commercialized kits by proficient groups is necessary before any recommendation can be made to parasitology laboratories by health authorities or learned societies. Here, we compared the performance of an original commercial method, the Iam TOXO Q-LAMP (DiaSorin®), using Loop-mediated isothermal amplification (LAMP) technology, with our reference laboratory-developed method using real-time PCR. The kit was first tested using amniotic fluid (AF) and plasma samples (either negative or spiked with live T. gondii tachyzoites at different concentrations (from 7 to 10 5  tachyzoites/mL)). It was then assessed using a cohort of 11 AF, five placental and 32 blood clinical samples preserved at −20 °C. For the processing of placental/blood samples, a pretreatment step was used, which did not strictly follow the manufacturer’s recommendations. The practical ease of use and compliance with good laboratory practices were also evaluated. Although the LAMP assay was less sensitive than the laboratory-developed method at very low parasite concentrations (0.1  T. gondii genome equivalents/mL), the two methods yielded identical results qualitatively and, in some instances, quantitatively, particularly for AF samples. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: To assess long‐term efficacy and safety of self‐made cryopreservative fibrin glue (SMC) applied in pterygium surgery. Methods: Prospective, comparative, interventional case series. Forty eyes of 40 patients with nasal primary pterygium, 24 male and 16 female, were enrolled. The patients were assigned to two groups and each contained 12 male and eight female based on the pterygium area encroaching onto the cornea. In one group, the conjunctival autograft was attached to the sclera with SMC stored for 2 months, and in the other group, commercial fibrin glue kit (CK) was applied after the pterygium was removed. All the patients were followed up postoperatively on days 1, 3, 7 and 14 then at months 1, 3, 6, 12. The main outcome measures included operating time, postoperative discomfort, recurrence rate and complications. Results: There were no significant differences in surgery time (p = 0.713) and postoperative discomfort (day 1, 3, 7; p = 0.747, p = 0.766, p = 0.983, respectively) between the two groups. By the end of 1‐year follow‐up, the recurrence rate was 0% in the SMC group and 5% in the CK group (p = 1.000). There were no infections and severe visual acuity (VA) threatening complications in either group. Conclusion: Self‐made cryopreservative fibrin glue (SMC) is as effective as standard CK for autograft fixation in pterygium surgery and it also has good safety after long‐term follow‐up. For its convenience and low cost, this new methods should be popularized, especially in underdeveloped area. [ABSTRACT FROM AUTHOR]

In this report, we describe the first national scale multi-laboratory evaluation of monkeypox virus (MPXV) DNA commercial PCR kits. The objective of this study was to evaluate 2 kits by different diagnostic laboratories across Israel. Ten standardized samples were tested simultaneously using the Novaplex (15 laboratories) and Bio-Speedy (seven laboratories) kits. An in-house assay based on previously published reactions was used as reference. Comparison of the results showed high intra-assay agreement between laboratories, with small variations for most samples. The in-house assay had an analytical detection limit of less than 10 copies per reaction. While the 2 commercial kits were able to detect specimens with low viral loads similarly to the in-house assay, significant differences were observed, in the Cq values and relative fluorescence (RF), between the assays. The RF signal of the in-house and Bio-Speedy assays ranged between 5,000 and 10,000 RFU, while the signal in the Novaplex assay was less than 600 RFU. Due to the kit measurement protocol, the Cq values of the Bio-Speedy kit were 5 to 7.5 cycles lower than those of the in-house assay. On the contrary, the Cq values of the Novaplex kit were significantly higher than those of the in-house assay, with differences of 3 to 5 cycles per sample. Our results suggest that while all assays were similar in their overall sensitivity, direct comparison of Cq values between them may be misleading. To our knowledge, this is the first methodical evaluation of commercial MPX test kits. We therefore anticipate that this study would help diagnostic laboratories in choosing a specific MPX detection assay. IMPORTANCE To the best of our knowledge, this study is the first methodical evaluation of commercial kits designed for Monkeypox virus detection. This was done by performing the same tests using the same sample set in multiple laboratories, simultaneously, on a national scale. It therefore provides important and unique information on the performance of such kits and provides a guideline for choosing the assay of choice for monkeypox virus diagnosis in a standard diagnostic laboratory. It also demonstrates potential complications when trying to compare the results of different assays, even when testing exactly the same samples, under identical conditions., Competing Interests: The authors declare no conflict of interest.

A global decline in pollinator abundance and diversity has demanded increased research attention to the ecology and genetics of bumblebees. However, as progressively more restrictions are placed on sampling for insects, researchers are increasingly obliged to use archival specimens collected for purposes other than genetic analyses. In this study we assessed the suitability, for population genetic studies, of popular, low-cost methods for preservation and storage of bumblebee specimens. Specimens of Bombus terrestris L. were held under six storage regimes for up to two years. DNA was extracted from the samples using three extraction protocols and the quality of the DNA was examined using PCR amplification of a mitochondrial and a nuclear gene. All extraction and storage methods provided sufficient DNA for successful PCR amplification. However, samples preserved in acetone or at freezing temperatures yielded the highest DNA concentrations. DNA yields from pinned specimens at room temperature declined over time, particularly when using standard extraction techniques. DNA concentrations were significantly lower from specimens preserved in 70% ethanol compared to all other extraction techniques and declined linearly over the two years of storage. These results indicate that two of the most popular insect storage methods (pinning and storage in ethanol) should be avoided for the long-term preservation of genetic material for future studies. We suggest that optimal insect preservation methods should be incorporated into research protocols in order to best capitalise on limited collection opportunities.

Simple, fast, low-cost and high throughput protocols are required for DNA isolation of plant species. In this study, phenol chloroform isoamyl alcohol and commercial (Sigma) DNA isolation kit methods were applied on some Inula species that belong to Asteraceae family. Genomic DNA amounts, A260, A280, A260/A230 and purity degrees (A260/A280) that were obtained through both methods were measured through electrophoresis and spectrophotometer. Additionally, PCR amplification was realized by primer pairs specific to nrDNA ITS, cpDNA ndhF (972F-1603R) and trnL-F regions. Results showed that maximum genomic DNA in nanograms obtained by phenol chloroform isoamyl alcohol method. The study also revealed that I. macrocephala had the maximum DNA and I. heterolepis had the minimum DNA amount. A260/A280 purity degrees showed that the highest and lowest purity in gDNAs obtained through phenol-chloroform isoamyl alcohol method were in I.aucheriana and I. salicina, respectively. The highest and lowest purity degrees of gDNAs obtained through commercial kit was observed in I. fragilis and I. macrocephala samples, respectively. PCR amplification results showed that while band profiles of each three regions (ITS, trnL-F and ndhF) did not yield positive results in PCR amplifications using phenol-chloroform isoamyl alcohol method; PCR band profiles obtained through commercial kit yielded positive results. As a result, it is fair to say that the relation of genomic DNA with PCR was found to be more efficient although the maximum amount of genomic DNA was obtained through phenol chloroform isoamyl alcohol method. [ABSTRACT FROM AUTHOR]

High quality, pure DNA is required for ensuring reliable and reproducible results in molecular diagnosis applications. A number of in-house and commercial methods are available for the extraction and purification of genomic DNA from faecal material, each one offering a specific combination of performance, cost-effectiveness, and easiness of use that should be conveniently evaluated in function of the pathogen of interest. In this comparative study the marketed kits QIAamp DNA stool mini (Qiagen), SpeedTools DNA extraction (Biotools), DNAExtract-VK (Vacunek), PowerFecal DNA isolation (MoBio), and Wizard magnetic DNA purification system (Promega Corporation) were assessed for their efficacy in obtaining DNA of the most relevant enteric protozoan parasites associated to gastrointestinal disease globally. A panel of 113 stool specimens of clinically confirmed patients with cryptosporidiosis ( n = 29), giardiasis ( n = 47) and amoebiasis by Entamoeba histolytica ( n = 3) or E. dispar ( n = 10) and apparently healthy subjects ( n = 24) were used for this purpose. Stool samples were aliquoted in five sub-samples and individually processed by each extraction method evaluated. Purified DNA samples were subsequently tested in PCR-based assays routinely used in our laboratory. The five compared methods yielded amplifiable amounts of DNA of the pathogens tested, although performance differences were observed among them depending on the parasite and the infection burden. Methods combining chemical, enzymatic and/or mechanical lysis procedures at temperatures of at least 56 °C were proven more efficient for the release of DNA from Cryptosporidium oocysts. [ABSTRACT FROM AUTHOR]