Cross-species amplification from non-human primate DNAs in commercial human DNA assay kits.

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.

Age- and sex-adjusted reference intervals for steroid hormones measured by liquid chromatography-tandem mass spectrometry using a widely available kit.

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.

Fast preparation of high-quality viral dsRNA from fungal tissue by commercial nucleic acid extraction kits.

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.

A quick, easy and efficient protocol for extracting high-quality RNA from Mycobacterium tuberculosis using a spin column commercial kit.

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.

A Multi-Laboratory Evaluation of Commercial Monkeypox Virus Molecular Tests.

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.

Comparative Evaluation of Three Commercial Quantitative Real-Time PCRs Used in Japan for Bovine Leukemia Virus.

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.

Characterization and Comparison of Mesenchymal Stem Cell-Derived Exosome Isolation Methods using Culture Supernatant.

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.

Serodiagnosis of canine leishmaniasis using a novel recombinant chimeric protein constructed with distinct B-cell epitopes from antigenic Leishmania infantum proteins.

Visceral leishmaniasis (VL) is an important public health problem in the world, and control measures are insufficient to avoid the spread of this neglected disease. Dogs are important domestic reservoirs of Leishmania parasites in countries where VL is a zoonosis, representing a major source of infection between sand fly vectors and humans. In this context, a precise diagnosis of canine leishmaniasis (CanL) could help to reduce the number of human cases. Distinct approaches for the diagnosis of CanL have used recombinant proteins in serological assays. However, variable results of the antigens have been found, mainly to diagnosis asymptomatic cases. The present study used bioinformatics to select specific B-cell epitopes of four Leishmania infantum proteins, which had previously been proven to be antigenic in VL, aiming to produce a novel chimeric protein and to evaluate it for the diagnosis of CanL. Seven B-cell epitopes were identified and used to construct the chimera, which was analyzed in a recombinant format through an ELISA assay against a canine serological panel. A soluble Leishmania antigenic extract (SLA) was used as an antigen control. Results showed 100 % sensitivity and specificity for chimera, while when using SLA the values were 26.0 % and 96.4 %, respectively. The performance of chimera was compared with a commercial kit using asymptomatic and symptomatic dog sera, and the data showed that no false-negative result was found when the recombinant protein was used. However, when using the commercial kit, 40.0 % and 16.0 % of the false-negative results were found, respectively. In conclusion, the recombinant chimera showed an antigenic potential to be evaluated in new studies against a larger serological panel for the diagnosis of CanL.

Advanced sequence optimization for the high efficient yield of human group A rotavirus VP6 recombinant protein in Escherichia coli and its use as immunogen.

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.

A Comparative Study of Some Procedures for Isolation of Fruit DNA of Sufficient Quality for PCR-Based Assays.

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.

Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions.

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.

Biases during DNA extraction affect characterization of the microbiota associated with larvae of the Pacific white shrimp, Litopenaeus vannamei.

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.

A new LAMP-based assay for the molecular diagnosis of toxoplasmosis: comparison with a proficient PCR assay.

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 105 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.

Self-made cryopreservative fibrin glue applied in pterygium surgery: a novel practical technique.

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.

Comparison of self-made cryopreservative fibrin glue and commercial fibrin glue kit in pterygium surgery: 1-year follow-up.

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.

Comparison of a commercial real-time PCR assay, RealCycler® PJIR kit, progenie molecular, to an in-house real-time PCR assay for the diagnosis of Pneumocystis jirovecii infections.

We compared the RealCycler® PJIR kit (Progenie Molecular), available in Europe, to an in-house real-time PCR assay for the diagnosis of Pneumocystis jirovecii infections. Excellent agreement was found (concordance rate, 97.4%; Cohen's kappa, 0.918>0.8) showing that this commercial assay represents an alternative method for the diagnosis of P. jirovecii infections.

Evaluation of five commercial methods for the extraction and purification of DNA from human faecal samples for downstream molecular detection of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Entamoeba spp.

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.