Multiple PCR Research Articles

An outbreak of tuberculosis in endangered northern pig-tailed macaques (Macaca leonina) and milu deer (Elaphurus davidianus) from a zoo in China.

Tuberculosis (TB) is a zoonotic disease that affects humans and domesticated and wild animals. Animals in zoos are potentially an important source of TB for humans; however they are often neglected in routine disease surveillance programs. This investigation reports an outbreak of TB in milu deer and northern pig-tailed macaques in a zoo in Wuhan, China, which highlighted the need for improved prevention and control of TB in China. Between 24 November and 9 December 2020 two milu deer and a northern pig-tailed macaque that were displaying signs of wasting died. Post-mortem, histopathological diagnosis and acid fast staining were used for the dead animals. Multiple PCR for Mycobacterium tuberculosis complex (MTBC) was performed to identify the bacterial in both milu deer and northern pig-tailed macaque. The serum antibody iELISA for MTBC was then performed for all the surviving milu deer and northern pig-tailed macaques. Six seropositive milu deer and a seropositive northern pig-tailed macaque were subsequently euthanised and, along with two other dead milu deer, necropsied. DNA from these tissue samples was extracted and detected MTBC using PCR and Real-time PCR. Subsequently bacterial isolation was used to confirm the infection. The lungs of the dead animals displayed gross and histological TB-like lesions and changes, and red staining bacilli were detected in smears of the lesions by microscopy after acid fast staining. Mycobacterium bovis (M. bovis) was detected in the two milu deer and Mycobacterium tuberculosis (M. tb) in the northern pig-tailed macaque using multiple PCR for MTBC. 35.3% surviving milu deer and 50% surviving northern pig-tailed macaques MTBC serologically positive. Six of the euthanised milu deer were also positive on a DNA test for M. bovis and the euthanised northern pig-tailed macaque was positive to M. tb. This is the first report of tuberculosis in the endangered species, milu deer and northern pig-tailed macaques, in China, and warrants urgent attention by researchers and conservation authorities. These cases highlight the need for expanding surveillance for MTBC to zoos in China.

Mapping of a QTL associated with sucrose content in peanut kernels using BSA-seq.

As an important factor affecting the edible quality of peanut kernels, sucrose content is a complex quantitative trait regulated by multiple factors. In this study, an F2 segregating population and a recombinant inbred line (RIL) population, derived from a cross between the high sucrose content variety Jihuatian 1 and the low sucrose content line PI478819, were used as materials to map a quantitative trait locus (QTL) associated with sucrose content in peanut kernels. Four QTLs were initially located on chromosomes A03 and A06 based on BSA-seq technology, and multiple kompetitive allele-specific PCR markers were developed based on single-nucleotide polymorphisms (SNPs) in the intervals. The markers were genotyped in the RIL population and finely mapped to a stable QTL, qSUCA06, located on chromosome A06 within a 0.29-Mb physical genomic interval (112367085-112662675 bp), which accounted for 31.95%-41.05% of the phenotypic variance explained. SNP and insertion/deletion annotations were performed on genes in the candidate interval, and having screened out those genes with mutations in exons, candidate genes were verified by qRT-PCR. The results revealed that Arahy.Y2LWD9 may be the main gene regulating sucrose content. The QTL identified in this study will not only contribute to marker-assisted breeding for improvement of peanut sucrose content but also paves the way for identifying gene function.

Coinfections with SARS-CoV-2 variants and influenza virus during the 2019 Coronavirus disease pandemic in Burkina Faso: A surveillance study.

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) particularly the variants of concern coinfections with influenza is a public health concern in Africa. We aimed to characterize the SARS-CoV-2 variants and determine the rate of coinfections with influenza in Burkina Faso. COVID-19 surveillance study was conducted between August 2021 and January 2022 using reverse transcription polymerase chain reaction(RT-PCR). Positive specimens were further screened for SARS-CoV-2 variants using the multiple variants real-time PCR kits. In addition, influenza virus strains were detected by RT-PCR in SARS-CoV-2 positive specimens using the CDC primers, probes, and protocols. Of 324 specimens assessed, the Omicron and Delta variants of SARS-CoV-2 were the most prevalent with 27.2% [95% confident interval (CI): 22.5-32.4] and 22.2% [95% CI: 17.9-27.2], respectively. The Beta and Gamma variants were detected in 4.3% [95% CI: 2.4-7.1] and 0.3% [95% CI: 0.0-1.7], respectively. Coinfections of Omicron and Beta variants were reported in 21.3% [95% CI: 17.0-26.2], Omicron and Delta variants in 1.2% [95% CI: 0.3-3.1] of specimens, and the Omicron-Gamma variants' coinfections in 0.6% [95% CI: 0.1-2.2]. One COVID-19 specimen with an undetected SARS-CoV-2 variant was also tested positive for the seasonal influenza A (H3N2) virus. No cases of pandemic influenza A (H1N1)pdm09, seasonal A/H1N1, and influenza B were detected. The current WorldHealth OrganizationSARS-CoV-2 variants of concern were prevalent and their coinfections with influenza were uncommon. Continuous surveillance of both pathogens is, however, needed because of their public health implications.

Real Time PCR-based diagnosis of human visceral leishmaniasis using urine samples.

Diagnosis of visceral leishmaniasis (VL) through the detection of its causative agents namely Leishmania donovani and L. infantum is traditionally based on immunochromatographic tests, microscopy of bone marrow, spleen aspirates, liver or lymph node and differential diagnosis. While the first process has low specificity, the later one carries the risk of fatal hemorrhage. Over the last decade, multiple Polymerase Chain Reaction (PCR) based diagnosis has been developed using blood and urine sample with a varying degree of sensitivity and specificity, an issue worth improving for precision diagnosis. Earlier, we reported a PCR-based diagnosis of L. donovani in peripheral blood using a novel set of PCR primers with absolute specificity. Using the same set of primers and PCR conditions, here we describe diagnosis of L. donovani from urine, for a non-invasive, rapid and safe diagnosis. Diagnosis of VL was carried out using urine samples collected from clinically diagnosed VL patients (n = 23) of Bangladesh in Real Time PCR. Test results were validated by comparing blood samples from the same set of patients. Sensitivity and specificity of this diagnosis was analyzed using retrospective bone marrow samples, collected earlier from confirmed VL patients (n = 19). The method showed 100% sensitivity in detecting L. donovani in urine and corresponding blood and retrospective bone marrow samples, as well as 100% specificity in control groups. A Real Time PCR-based molecular detection system using urine sample is hereafter presented what could be a, non-invasive approach for VL detection with precision and perfection.

631. CMV Reactivation in Hematopoietic Cell Transplant Candidates: A Novel Risk Factor for Post-transplant Reactivation

Abstract Background CMV reactivation is occasionally detected during the work-up for hematopoietic cell transplantation (HCT) but its natural history and significance on posttransplant CMV risk is unknown. Methods CMV seropositive 1st HCT allograft recipients in the preemptive therapy era (2010-17) were analyzed. CMV PCR testing was routinely performed before and after HCT. Cumulative incidences of CMV reactivation after HCT were calculated at multiple PCR thresholds by pre-HCT viral loads (peak: < or >150 IU/mL; last prior to HCT positive vs positive to negative). Treatment of pre-HCT reactivation was recommended for viral loads >50 IU/mL. Multivariable Cox proportional hazard models were used to determine the association of pre-transplant CMV reactivation on post-HCT CMV reactivation at different PCR thresholds. Results Among 1536 patients (median age 50 y, range 0.1-81), 155 (10%), 53 (3.5%) and 24 (1.6%) had pre-HCT reactivation in the month before HCT at any level, >150 IU/mL, and >500 IU/mL, respectively. Pre-transplant CMV reactivation was associated with a higher risk of post-HCT CMV reactivation at all examined PCR thresholds and with CMV disease after transplantation in cumulative incidence (Figure 1) and multivariable analyses (Figure 2A). Patients who had CMV DNA detected that subsequently became negative before HCT (with or without treatment) had a lower risk than those who proceeded to HCT with viremia (Figure 2B). Cumulative Incidence of CMV Infection and Disease Multivariable Cox Regression Conclusion CMV reactivation occurs pre-HCT in ∼10% of CMV seropositive HCT candidates and is a risk factor for post-HCT CMV events at all severity levels, placing a HCT recipient in a high-risk category. Thus, pre-HCT PCR testing and antiviral treatment as well as the use of optimized posttransplant antiviral or immunotherapy prevention strategies are recommended. Disclosures Joshua A. Hill, MD, Allovir: Advisor/Consultant|Allovir: Grant/Research Support|Covance/CSL: Advisor/Consultant|CRISPR: Advisor/Consultant|Deverra: Grant/Research Support|Gilead: Grant/Research Support|Karius: Advisor/Consultant|Karius: Grant/Research Support|Merck: Grant/Research Support|Octapharma: Advisor/Consultant|OptumHealth: Advisor/Consultant|Oxford Immunotec: Grant/Research Support|Pfizer: Advisor/Consultant|Symbio: Advisor/Consultant|Takeda: Advisor/Consultant Leona Holmberg, MD, PhD, Bristol Myers Squibb: Grant/Research Support|Janssen: Grant/Research Support|Merck: Grant/Research Support|Millennium-Takada: Grant/Research Support|Sanofi: Grant/Research Support|Seattle Genetics: Grant/Research Support|Up to Date: Royalty Alpana Waghmare, MD, Allovir: Grant/Research Support|Ansun Biopharma: Grant/Research Support|Devarra Therapeutics: DSMB|Kyorin Pharmaceutical: Advisor/Consultant|Pfizer: Grant/Research Support|Vir/GSK: Grant/Research Support Alexander L. Greninger, MD, PhD, Abbott: Contract Testing|Cepheid: Contract Testing|Gilead: Grant/Research Support|Gilead: Contract Testing|Hologic: Contract Testing|Merck: Grant/Research Support|Novavax: Contract Testing|Pfizer: Contract Testing Mary Flowers, MD, Incyte Corp.: Grant/Research Support|Janssen: Honoraria|Johnson & Johnson: Honoraria|Novartis: Honoraria|Pharmacyclics, Inc.: Grant/Research Support Geoffrey Hill, M.D., FRACP, FRCPA, Applied Molecular Transport: Grant/Research Support|Compass Therapeutics: Grant/Research Support|Generon Corporation: Advisor/Consultant|Heat Biologics: Grant/Research Support|iTeos Therapeutics: Advisor/Consultant|iTeos Therapeutics: Grant/Research Support|Laevoroc Oncology: Grant/Research Support|NapaJen Pharma: Advisor/Consultant|Neoleukin Therapeutics: Advisor/Consultant|Serplus Technology: Grant/Research Support|Syndax Pharmaceuticals: Grant/Research Support Michael J. Boeckh, MD PhD, Allovir: Advisor/Consultant|Amazon: Grant/Research Support|Ansun Biopharma: Grant/Research Support|EvrysBio: Advisor/Consultant|Gates Ventures: Grant/Research Support|Gilead Sciences: Advisor/Consultant|Gilead Sciences: Grant/Research Support|GlaxoSmithKline: Advisor/Consultant|GlaxoSmithKline: Grant/Research Support|Helocyte: Advisor/Consultant|Janssen: Advisor/Consultant|Janssen: Grant/Research Support|Kyorin Pharmaceuticals: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Moderna: Advisor/Consultant|Moderna: Grant/Research Support|Regeneron: Grant/Research Support|ReViral: Advisor/Consultant|Symbio: Advisor/Consultant|Takeda: Grant/Research Support|Vir Biotechnology: Advisor/Consultant|Vir Biotechnology: Grant/Research Support.

High-throughput primer design by scoring in piecewise logistic model for multiple polymerase chain reaction variants

Polymerase chain reaction (PCR) variants requiring specific primer types are widely used in various PCR experiments, including generic PCR, inverse PCR, anchored PCR, and ARMS PCR. Few tools can be adapted for multiple PCR variants, and many tools select primers by filtration based on the given parameters, which result in frequent design failures. Here we introduce PrimerScore2, a robust high-throughput primer design tool that can design primers in one click for multiple PCR variants. It scores primers using a piecewise logistic model and the highest-scored primers are selected avoiding the issue of design failure and the necessity to loosen parameters to redesign, and it creatively evaluates specificity by predicting the efficiencies of all target/non-target products. To assess the prediction accuracy of the scores and efficiencies, two next generation sequencing (NGS) libraries were constructed—a 12-plex and a 57-plex—and the results showed that 17 out of 19 (89.5%) low-scoring pairs had a poor depth, 18 out of 19 (94.7%) high-scoring pairs had a high depth, and the depth ratios of the products were linearly correlated with the predicted efficiencies with a slope of 1.025 and a coefficient of determination (R2) 0.935. 116-plex and 114-plex anchored PCR panels designed by PrimerScore2 were applied to 26 maternal plasma samples with male fetuses, the results showed that the predicted fetal DNA fractions were concordant with fractions measured in gold standard method (Y fractions). PrimerScore2 was also used to design 77 monoplex Sanger sequencing primers, the sequencing results indicated that all the primers were effective.

Analysis of non-bacterial respiratory pathogens in children in Ningbo City from 2019 to 2021

Objective: To investigate the distribution characteristics of respiratory non-bacterial pathogens in children in Ningbo from 2019 to 2021. Methods: A retrospective analysis was performed on 23 733 children with respiratory tract infection who visited the department of pediatrics of Ningbo Women and Children's Hospital from July 2019 to December 2021. There were 13 509 males (56.92%) and 10 224 females (43.08%), with an age range of 1 day to 18 years old. There were 981 cases in the neonatal group (younger than 1 month old), 5 880 cases in the infant group (1 month to younger than 1 year old), 6 552 cases in the toddler group (1 to younger than 3 years old), 7 638 cases in the preschool group (3 to younger than 7 years old), and 2 682 cases in the school-age group (7 to 18 years old). Thirteen respiratory pathogens were detected by multiple polymerase chain reaction (PCR) based on capillary electrophoresis, and SPSS 23.0 software was used for statistical analysis of the results, the count data were expressed as percentages, and the χ2 test was used for comparison between groups. Results: Of the 23 733 specimens, 13 330 were positive for respiratory pathogens, with a total positive rate of 56.17%. The positive rates of human rhinovirus (HRV) 24.05% (5 707/23 733), human respiratory syncytial virus (HRSV) 10.45% (2 480/2 3733) and mycoplasma pneumoniae (Mp) 7.03% (1 668/23 733) were in the first three. The positive rates of pathogens in the male and female children were 57.47% (7 763/13 509) and 54.45% (5 567/10 224), respectively, and the difference was statistically significant (χ2=21.488, P<0.001). The positive rates in the neonatal group, infant group, toddler group, preschool group, and school-age group were 31.80% (312/981), 54.71% (3 217/5 880), 63.23% (4 143/6 552), 59.83% (4 570/7 638), 40.57% (1 088/2 682), respectively, and the difference among the groups was statistically significant (χ2=681.225, P<0.001). The single infection rate was 47.43% (11 256/23 733), the mixed infection rate of two or more pathogens was 8.74% (2 074/23 733), most of which were mixed infections of two pathogens. HRV, HADV, HCOV, Ch disseminated in the whole year. HRSV, HMPV, Boca, HPIV occurred mostly in fall and winter. The positive rates of FluA, FluB, Mp were at a low level after the corona virus disease 2019 (COVID-19) epidemic (2020 and 2021). The positive rates of FluA, H1N1, H3N2, FluB, HADV, Mp in 2020 were significantly lower than in 2019 (P<0.05). The positive rates of HPIV, HRV, HCOV, Ch in 2020 were significantly higher than in 2019 (P<0.05). The positive rates of FluA, H1N1, H3N2, HPIV, HCOV, Mp, Ch in 2021 were significantly lower than in 2020 (P<0.05). The positive rates of Boca, HMPV, HRSV in 2021 were significantly higher than in 2020 (P<0.05). Conclusion: From 2019 to 2021, the main non-bacterial respiratory pathogens of children in Ningbo City were Mp and HRV, and the detection rates of respiratory pathogens varied among different ages, seasons and genders.

TYK2 correlates with immune infiltration: A prognostic marker for head and neck squamous cell carcinoma.

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family and is involved in immune and inflammatory signaling. TYK2 is overexpressed in several types of cancers and promotes the invasion and proliferation of cancer cells. Nevertheless, the roles of TYK2 in the prognosis and immune infiltration of head and neck squamous cell carcinoma (HNSCC) remain to be elucidated. In this study, the expression of TYK2 in HNSCC was evaluated based on the data retrieved from multiple databases and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The prognostic potential of TYK2 in patients with HNSCC was analyzed by Kaplan-Meier curves and Cox regression analysis. A TYK2-related risk assessment model was subsequently constructed by Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis and stepwise multivariate Cox regression analysis. The association between the expression of TYK2 and the tumor immune microenvironment, immune checkpoints, and drug sensitivity was explored various packages in R. Cell function assays were finally used for exploring the effects of TYK2 on the growth and metastasis of HNSCC tumors. The expression of TYK2 was significantly upregulated in HNSCC and was found to be closely correlated with HPV status, gender, clinical grade, and TP53 mutation status. Survival analysis suggested that TYK2 is associated with better survival outcomes and acts as an independent prognostic indicator of HNSCC. The model constructed herein also performed well in terms of predicting patient prognosis. The expression of TYK2 was positively associated with the population of tumor-infiltrating immune cells, expression of immune checkpoint genes, and antitumor drug susceptibility. Functionally, TYK2 knockdown significantly promoted the proliferation, migration, and invasion of HNSCC cell lines in vitro. The findings demonstrated that TYK2 could serve as a suppressor of tumor growth and holds significant promise as a novel biomarker for assessing the prognosis of patients with HNSCC and aid in immunotherapy against HNSCC.

Universal hydrolysis probe-based approach for specific detection and genotyping of foodborne pathogens

Real-time PCR assays are the method of choice for the specific detection of DNA targets. Multiple real-time PCR chemistries are used for developing pathogen detection assays. Among them, a hydrolysis probe is a preferred choice for pathogen detection assays. Two known limitations of hydrolysis probes are high cost and limited storage life. Therefore, this study aimed to develop and validate a universal hydrolysis probe (UHP)-based approach with high-resolution melt (HRM) analysis capabilities. The approach can be used for the detection and genotyping of target DNA. The approach described in this study was validated by standardizing nine UHP assays for detecting seven Shiga toxin-producing Escherichia coli serogroups, Listeria monocytogenes, and Salmonella strains. These nine assays were validated with 141 pure culture bacterial strains. Additionally, the HRM capability of the developed approach was validated for three UHP assays targeting E. coli O26, O111, and O121 using 96 DNAs isolated from enriched food samples. The nine assays specifically detected the target bacterial strains, and the three assays showed single nucleotide polymorphism (SNP) identification capability and no cross-reactivity with non-target strains. The developed approach can be performed in singleplex or multiplex format and combined with HRM analysis. The data from this study demonstrate that the UHP real-time PCR approach is a robust method for detecting any deoxyribonucleic acid target.

Isolation of Escherichia coli and Klebsiella pneumoniae strains producing extended spectrum β-lactamases from dog urine of the Metropolitan Area of the Aburrá Valley Antioquia-Colombia

Escherichia coli and Klebsiella pneumoniae are the most common pathogens causing urinary tract infections in humans and animals. Close contact between humans and companion animals can facilitate the spread of multidrugresistant pathogens between both species. Objective: To characterize extended-spectrum β-lactamases (ESBL) -producing E. coli and K. pneumoniae isolated from dogs with urinary tract infections in the metropolitan area of Valle del Aburrá (Antioquia, Colombia). Methods: Three-hundred seventy-one urine samples collected from March 2018 to March 2019 in a veterinary clinical laboratory were analyzed. E. coli and K. pneumoniae isolates were detected in chromogenic agar and identified by biochemical tests. Susceptibility testing was performed by disc diffusion and ESBL production was evaluated by the double disk test in all isolates. MIC determination of ESBL-positive isolates were performed on the automated VITEK®2 system. Multiple PCR was used for the detection of CTX-M beta-lactamases (group 1, 2, 9 and 8/25), SHV, TEM and AmpC of plasmid origin in ESBL-positive isolates. Results: In total 22 out 371 isolates were positive for ESBL production by double disc test, 11 E. coli (ESBL-Ec) and 11 K. pneumoniae (ESBL-Kp). The multiple PCR detected CTX-M group 1 in the 22 ESBL-positive isolates. Multi-drug resistance was observed in all ESBL-producing isolates Conclusions: A high frequency of antibiotic multi-resistance was found in ESBL-Ec and ESBL-Kp. The main ESBL detected was CTX-M group 1, which also prevails in human isolates.

A multiplex PCR-based sensitive and specific method for detecting Y chromosome material in patients with Turner syndrome

To develop a multiplex PCR method for a rapid detection of Y chromosome-specific sequences in patients with Turner syndrome. Nine genes were selected from various regions of the Y chromosome for designing the primers, which included SRY, TBL1Y, TSPY on the short arm of the Y chromosome, DDX3Y, HSFY1, RPS4Y2 and CDY1 on the long arm of Y chromosome and SHOX in the short arm and SPRY3 in the long arm of the pseudoautosomal region (PAR) of X and Y chromosomes. A multiplex PCR method for the nine genes in Y chromosome was established and optimized. The sensitivity was tested by using different amounts of genomic DNA. A total of 36 patients with Turner syndrome and a patient with male dwarfism with karyotype of 46, X, +mar were examined by the multiplex PCR method for the existence of materials from the Y chromosome. The optimization results of the multiplex PCR reaction system (50 μL) showed that when the final concentration of upstream and downstream of each pair of primers was 0.1 μM, the multiplex PCR reaction of the 9 pairs of primers clearly amplified the target with the expected band size, and there was no non-specific amplification. The bands were clearly visible when the amount of genomic DNA in the multiple PCR reaction system was as low as 1 ng. By using the method, we have examined the 36 patients with Turner syndrome. One patient with Turner syndrome with karyotype of 45,X[40]/47XYY[21] amplified specific seven genes on Y chromosome, 35 patients with Turner syndrome amplified only two target genes SHOX and SPRY3, but not the other seven specific genes on the Y chromosome, which was in keeping with the clinical manifestations of such patients. This study established a multiplex PCR reaction system with nine genes, which can quickly and accurately screen Y chromosome materials in patients with Turner syndrome. It has the advantages of low cost, simple operation, high specificity and rapid turn-around time, and can be used to detect Turner syndrome patients with Y chromosome material in time. The method has provided a diagnostic basis for preventive gonad resection to prevent malignant gonadal tumors.

A SEVERE ADVERSE DRUG REACTION (ADR) TO REGEN-COV (CASIRIVIMAB AND IMDEVIMAB)

<h3>Introduction</h3> A promising means to limit the progression of the SARS-CoV-2 virus is the use of monoclonal antibodies (mAbs), which are able to recognize viral antigenic epitopes of the Spike protein. Despite their high degree of efficacy, mAbs are sometimes accompanied by adverse drug reactions (ADRs) and although rare, they do occur and their use should be carefully determined by risk benefit considerations. <h3>Case Description</h3> A 33-year-old female with a history of intermittent asthma presented with a 7-day history of fatigue, myalgia, postnasal drip, diffuse palpable lymphadenopathy, and severe headache. During the first five days of her illness, she had multiple negative COVID-19 PCR and antigen tests. PCR test became positive on day 6 and symptoms had begun slowly improving on day 8. Because of slow improvement of symptoms, she was given IV dose of REGEN-COV(600 mg casirivimab, 600 mg imdevimab). About 12 hours after infusion, symptoms of fatigue and myalgias increased dramatically. For the next 5 days she experienced severe, diffuse and profound muscle tenderness, arthralgias, and fatigue. Symptoms gradually dissipated two weeks after the start of the mAb treatment. <h3>Discussion</h3> The unusual worsening of symptoms after the start of the mAb raises the question of a severe infusion-related reaction to REGEN-COV consisting of severe worsening of symptoms, increased arthralgias and diffuse muscle tenderness. Although the cause of these drug-related adverse reactions is unclear, the delayed start of the mAb may have contributed. In general, mAbs are usually safe but individual risks need to be carefully weighed against their expected therapeutic benefit.

Genomic selection of orange-spotted grouper (Epinephelus coioides) based on multiplex PCR enrichment capture sequencing

Orange spotted grouper (Epinephelus coioides) is an important mariculture fish, and genomic breeding of this grouper species has been hindered due to lack of efficient genotyping tools. Here, we developed a single nucleotide polymorphism (SNP) genotyping technology based on multiplex PCR enrichment capture sequencing, which mainly aims at target area for high-throughput sequencing, and 741 SNPs were designed for genomic selection (GS) of growth and ammonia tolerance traits at the same time. The multiplex PCR enrichment capture sequencing assay showed that the genotyping efficiency was more than 99% in the orange-spotted grouper and the predictive accuracy of body weight and ammonia tolerance traits was 82% and 96%, respectively. More importantly, the average identity of the sequences with these SNPs aligned to the genomes of giant grouper (E. lanceolatus) and brown-marbled grouper (E. fuscoguttatus) were both over 96%. Test data showed that the SNP genotyping efficiency was more than 94% in both giant grouper and brown-marbled grouper. In summary, these results indicated that the development of SNP loci and genotyping approach based on the multiple PCR enrichment capture sequencing are suitable for GS of growth and ammonia tolerance traits in various grouper species, and it would provide technical support for practical grouper breeding.

Correlation research of susceptibility single nucleotide polymorphisms and the severity of clinical symptoms in attention deficit hyperactivity disorder.

ObjectiveTo analyze the correlation between susceptibility single nucleotide polymorphisms (SNPs) and the severity of clinical symptoms in children with attention deficit hyperactivity disorder (ADHD), so as to supplement the clinical significance of gene polymorphism and increase our understanding of the association between genetic mutations and ADHD phenotypes.Methods193 children with ADHD were included in our study from February 2017 to February 2020 in the Children’s ADHD Clinic of the author’s medical institution. 23 ADHD susceptibility SNPs were selected based on the literature, and multiple polymerase chain reaction (PCR) targeted capture sequencing technology was used for gene analysis. A series of ADHD-related questionnaires were used to reflect the severity of the disease, and the correlation between the SNPs of specific sites and the severity of clinical symptoms was evaluated. R software was used to search for independent risk factors by multivariate logistic regression and the “corplot” package was used for correlation analysis.ResultsAmong the 23 SNP loci of ADHD children, no mutation was detected in 6 loci, and 2 loci did not conform to Hardy-Weinberg equilibrium. Of the remaining 15 loci, there were 9 SNPs, rs2652511 (SLC6A3 locus), rs1410739 (OBI1-AS1 locus), rs3768046 (TIE1 locus), rs223508 (MANBA locus), rs2906457 (ST3GAL3 locus), rs4916723 (LINC00461 locus), rs9677504 (SPAG16 locus), rs1427829 (intron) and rs11210892 (intron), correlated with the severity of clinical symptoms of ADHD. Specifically, rs1410739 (OBI1-AS1 locus) was found to simultaneously affect conduct problems, control ability and abstract thinking ability of children with ADHD.ConclusionThere were 9 SNPs significantly correlated with the severity of clinical symptoms in children with ADHD, and the rs1410739 (OBI1-AS1 locus) may provide a new direction for ADHD research. Our study builds on previous susceptibility research and further investigates the impact of a single SNP on the severity of clinical symptoms of ADHD. This can help improve the diagnosis, prognosis and treatment of ADHD.

Frequency and clinical impact of WT1 mutations in the context of CEBPA-mutated acute myeloid leukemia

ABSTRACT Introduction Several studies have confirmed that mutations in the Wilms tumor 1 (WT1) gene occur in adult acute myeloid leukemia (AML). However, few data are available regarding the incidence of WT1 mutations in CEBPAmut AML and their impact. Methods We retrospectively analyzed the frequency and clinical impact of WT1 mutations in 220 newly diagnosed AML patients with CEBPA mutations(CEBPAmut). Chromosome karyotype analysis was performed by R or G banding method and further confirmed either by fluorescence in situ hybridization (FISH) and/or by multiple reverse transcription polymerase chain reaction (multiple RT–PCR). Mutations were detected with a panel of 112mutational genes using next-generation sequencing (NGS). Results Overall, 30 WT1 mutations were detected in 29 of the 220 CEBPAmut AML patients (13.18%) screened. These mutations clustered overwhelmingly in exon 7 (n=16). WT1 mutations were found to be significantly more frequent in AML patients with double-mutated CEBPA (CEBPAdm) than in AML patients with single-mutated CEBPA (17.36%vs. 8.08%, P = 0.043). Among WT1-mutated patients, the most common co-mutation was FLT3-ITD (n = 7, 24.14%), followed by NRAS (n = 5, 17.24%), CSF3R (n = 4, 13.79%), GATA2 (n = 4, 13.79%), and KIT (n = 4, 13.79%). The most frequent functional pathway was signaling pathways inas many as 62.07% of cases. Notably,the concomitant mutations in epigenetic regulatorswere inversely correlated with WT1 mutations(P = 0.003). CEBPAdm AML patients with WT1 mutations had inferior relapse-free survival, event-free survival and overall survival compared with patients CEBPAdm AML without WT1 mutations (P = 0.002, 0.004, and 0.010, respectively). Conclusion Our data showed that WT1 mutations are frequently identified in CEBPAmut AML, especially in CEBPAdm AML. CEBPAmut AML patients with WT1 mutations show distinct spectrum of comutations. In the context of CEBPAdm AML, WT1 mutations predict a poor prognosis.

Single-tube Multiplex Nested PCR System for Efficient Detection of Pathogenic Microorganisms in SPF Rodents.

PCR testing is increasingly important for microbial control in SPF facilities. However, most current PCR methods are timeconsuming and require compromise between high sensitivity and high multiplexing. We developed a one-tube multiplex nested PCR strategy (MN-PCR) for simultaneous direct (that is, without culturing) detection of multiple pathogens. We first aligned sequences for the 16S rDNA genes of selected target bacteria and a panel of closely related organisms. From these data, we designed a pair of universal primers and multiple sets of species-specific PCR primers to amplify the target sequences; the universal primers were modified to include various degenerate bases and locked nucleic acids. In a single tube, 16S rDNA sequences were amplified by using the nested PCR primers under high temperature (that is, above 65°C) during the first stage of the MN-PCR procedure, when the target-species-specific PCR primers do not support amplification due to their short length. In addition, the concentration of the nested PCR primers during the first stage was adjusted to ensure that they were consumed and did not yield visible bands themselves. During the second stage, the enriched 16S rDNA sequences then served as templates for amplification of the species-specific fragments by using the multiple PCR primers at low annealing temperatures (that is, below 60°C). The results showed that our MN-PCR method detected as little as 1 fg of target bacterial DNA in a 20-μL reaction volume, whereas conventional multiplex PCR detected a minimum of 1 pg only. Compared with traditional multiplex PCR assays, our MN-PCR system is an effective and efficient culture-free process.

Dead migrants in the Mediterranean: genetic analysis of bone samples exposed to seawater

In April 2015, a fishing boat that departed from Libya with about 1,000 migrants on board sank in the Mediterranean Sea. Most of the migrants were packed in the hull of the boat and drowned in the shipwreck. After fifteen months, the ship was recovered from the seabed and brought to a Sicilian naval area for forensic investigations. Skeletal remains belonging to more than 700 people were retrieved. A selected sample composed of 80 victims was considered in order to evaluate the possibility of achieving genetic profiles useful for a positive identification from these challenging specimens. The molecular features of the DNA recovered from a significant number of real casework samples exposed to seawater for long periods of time were described for the first time. Three different DNA extraction protocols and three different commercial kits were employed in order to generate genetic profiles based on the characterization of 21 autosomal STR loci. The combination of multiple DNA extractions and the cross-checking of multiple PCR amplifications with different kits allowed to obtain reliable genetic profiles characterized by at least 16 STR markers in more than 70% of the samples. The factors that could have affected the different quality of the genetic profiles were investigated and the bone preservation was examined through microscopic and macroscopic analyses. The approach presented in this study could be useful in the management of the genetic analysis of bone samples collected in other similar DVI scenarios. The genetic profiles recovered from the bone samples will be compared in kinship analysis to putative relatives of the victims collected in Africa in order to obtain positive identifications.

Arabinosyltransferase C Mediates Multiple Drugs Intrinsic Resistance by Altering Cell Envelope Permeability in Mycobacterium abscessus.

ABSTRACTMycobacterium abscessus is an emerging human pathogen leading to significant morbidity and even mortality, intrinsically resistant to almost all the antibiotics available and so can be a nightmare. Mechanisms of its intrinsic resistance remain not fully understood. Here, we selected and confirmed an M. abscessus transposon mutant that is hypersensitive to multiple drugs including rifampin, rifabutin, vancomycin, clofazimine, linezolid, imipenem, levofloxacin, cefoxitin, and clarithromycin. The gene MAB_0189c encoding a putative arabinosyltransferase C was found to be disrupted, using a newly developed highly-efficient strategy combining next-generation sequencing and multiple PCR. Furthermore, selectable marker-free deletion of MAB_0189c recapitulated the hypersensitive phenotype. Disruption of MAB_0189c resulted in an inability to synthesize lipoarabinomannan and markedly enhanced its cell envelope permeability. Complementing MAB_0189c or M. tuberculosis embC restored the resistance phenotype. Importantly, treatment of M. abscessus with ethambutol, a first-line antituberculosis drug targeting arabinosyltransferases of M. tuberculosis, largely sensitized M. abscessus to multiple antibiotics in vitro. We finally tested activities of six selected drugs using a murine model of sustained M. abscessus infection and found that linezolid, rifabutin, and imipenem were active against the MAB_0189c deletion strain. These results identified MAB_0189 as a crucial determinant of intrinsic resistance of M. abscessus, and optimizing inhibitors targeting MAB_0189 might be a strategy to disarm the intrinsic multiple antibiotic resistance of M. abscessus.IMPORTANCE Mycobacterium abscessus is intrinsically resistant to most antibiotics, and treatment of its infections is highly challenging. The mechanisms of its intrinsic resistance remain not fully understood. Here we found a transposon mutant hypersensitive to a variety of drugs and identified the transposon inserted into the MAB_0189c (orthologous embC coding arabinosyltransferase, EmbC) gene by using a newly developed rapid and efficient approach. We further verified that the MAB_0189c gene played a significant role in its intrinsic resistance by decreasing the cell envelope permeability through affecting the production of lipoarabinomannan in its cell envelope. Lastly, we found the arabinosyltransferases inhibitor, ethambutol, increased activities of nine selected drugs in vitro. Knockout of MAB_0189c made M. abscessus become susceptible to 3 drugs in mice. These findings indicated that potential powerful M. abscessus EmbC inhibitor might be used to reverse the intrinsic resistance of M. abscessus to multiple drugs.

Skimming for barcodes: rapid production of mitochondrial genome and nuclear ribosomal repeat reference markers through shallow shotgun sequencing.

DNA barcoding is critical to conservation and biodiversity research, yet public reference databases are incomplete. Existing barcode databases are biased toward cytochrome oxidase subunit I (COI) and frequently lack associated voucher specimens or geospatial metadata, which can hinder reliable species assignments. The emergence of metabarcoding approaches such as environmental DNA (eDNA) has necessitated multiple marker techniques combined with barcode reference databases backed by voucher specimens. Reference barcodes have traditionally been generated by Sanger sequencing, however sequencing multiple markers is costly for large numbers of specimens, requires multiple separate PCR reactions, and limits resulting sequences to targeted regions. High-throughput sequencing techniques such as genome skimming enable assembly of complete mitogenomes, which contain the most commonly used barcoding loci (e.g., COI, 12S, 16S), as well as nuclear ribosomal repeat regions (e.g., ITS1&2, 18S). We evaluated the feasibility of genome skimming to generate barcode references databases for marine fishes by assembling complete mitogenomes and nuclear ribosomal repeats. We tested genome skimming across a taxonomically diverse selection of 12 marine fish species from the collections of the National Museum of Natural History, Smithsonian Institution. We generated two sequencing libraries per species to test the impact of shearing method (enzymatic or mechanical), extraction method (kit-based or automated), and input DNA concentration. We produced complete mitogenomes for all non-chondrichthyans (11/12 species) and assembled nuclear ribosomal repeats (18S-ITS1-5.8S-ITS2-28S) for all taxa. The quality and completeness of mitogenome assemblies was not impacted by shearing method, extraction method or input DNA concentration. Our results reaffirm that genome skimming is an efficient and (at scale) cost-effective method to generate all mitochondrial and common nuclear DNA barcoding loci for multiple species simultaneously, which has great potential to scale for future projects and facilitate completing barcode reference databases for marine fishes.

Analysis of relationship between distribution of pathogenic microorganisms in olfactory cleft and olfactory disorders among patients with upper respiratory inflammation during the prevention and control of COVID-19

Objective: To explore the relationship between pathogens in the olfactory cleft area and olfactory disorders in patients with upper respiratory inflammation (URI) during the prevention and control of 2019 novel coronavirus disease (COVID-19). Methods: A total of 234 URI patients including acute upper respiratory infection, chronic rhinosinusitis (CRS), allergic rhinitis (AR) were continuously selected from September 2020 to March 2021 in Beijing Anzhen Hospital and 98 healthy adults were enrolled as controls. The secretions from the olfactory cleft of all subjects were collected with nasal swabs under nasal endoscopy. Multiple real-time fluorescent quantitative polymerase chain reaction detection method was used to detect nucleic acids of 33 types of respiratory pathogenic microorganism. Sniffin' Sticks olfactory test was performed on all patients with URI. URI patients with olfactory dysfunction were followed up for 9 (8, 10) months (M (Q1, Q3)). SPSS 20.0 software was used for statistical analysis. Results: Among the 98 controls, 9 (9.18%) were positive for pathogenic microorganisms, including 1 (1.02%) rhinovirus, 1 (1.02%) parainfluenza virus type 3, 3 (3.06%) enterovirus, 1 (1.02%) staphylococcus aureus and 3 (3.06%) Moraxella catarrhalis. Among the 234 URI patients, 111 (47.44%) had olfactory disorders and 123 (52.56%) had normal sense of smell. In the olfactory disorder group (111 cases), 38 cases (34.23%) were positive for pathogenic microorganisms, and 4 cases (3.60%) were mixed infection, including 11 cases of rhinovirus (9.91%), 5 cases of coronavirus 229E (4.50%), 2 cases of coronavirus OC43/NL63 (1.80%), 3 cases of parainfluenza virus type 1 (2.70%), 2 cases of enterovirus (1.80%), 1 case of influenza B virus type BV (0.90%), 11 cases of Staphylococcus aureus (9.91%), 7 cases of Moraxella catarrhalis (6.31%), and 1 case of Klebsiella pneumoniae (0.90%). In the normal smell group (123 cases), 18 cases (14.63%) were positive for pathogenic microorganisms, and 1 case (0.81%) was mixed infection, including 3 cases of rhinovirus (2.44%), 4 cases of coronavirus 229E (3.25%), 1 case of Influenza virus type 3 (0.81%), 3 cases of enterovirus (2.44%), 3 cases of Staphylococcus aureus (2.44%), 4 cases of Moraxella catarrhalis (3.25%), and 1 case of Klebsiella pneumoniae (0.81%). Univariate analysis between the two groups found that there were significant differences in the detection rate of pathogenic microorganisms, rhinovirus and Staphylococcus aureus between the groups (all P<0.05). The detection rate of parainfluenza virus type 1, Staphylococcus aureus, and rhinovirus were different between the patients with olfactory disorder and normal olfactory function in the three subgroups of acute upper respiratory tract infection, CRS and AR, respectively (χ2 value was 3.88, 4.53 and 4.73, respectively, all P<0.05). During the follow-up period, among the 111 patients with olfactory disorder, 71 (63.96%) patients' olfactory function returned to normal, 32 (28.83%) patients' olfactory function improved but not completely returned to normal, 8 (7.21%) patients' olfactory function did not improve. Conclusions: During the prevention and control of COVID-19, rhinovirus or Staphylococcus aureus infection or colonization of URI patients is closely related to olfactory disorders. Parainfluenza virus type 1 infection can cause relatively persistent olfactory disorders in patients with acute upper respiratory tract infection. Staphylococcus aureus and rhinovirus colonization are related to the occurrence of olfactory dysfunction in CRS and AR patients respectively.