PCR Screening Research Articles

Evaluation of the Marine Bacterial Population in the Great Bitter Lake, Egypt, as a Source of Antimicrobial Secondary Metabolites

The ecological uniqueness of the Great Bitter Lake ecosystem makes its bacterial population interesting for investigation. Here, we present the first trial to evaluate the biosynthetic capacity of the bacterial population at the lake as a source of novel antimicrobials. We collected different samples from various locations throughout the lake including the oxic sediment, anoxic sediment, shore water, and off-shore water. We modified a molecular approach to compare and choose the samples with the highest bacterial biosynthetic capacity by quantifying the polyketide synthase gene clusters in their total community DNA. Furthermore, we screened the bacterial isolates recovered from these samples and their metabolic extracts for antimicrobial activity. We tried to tentatively investigate the identity of the active metabolites by PCR screening and LC–MS. The bacterial population in the oxic sediment had the highest biosynthetic capacity compared to other sample types. Four active Bacillus isolates were identified. The isolated Bacillus species were expected to produce numerous probable bioactive metabolites encoded by biosynthetic gene clusters related to the polyketide synthases (either individual or hybrid with non-ribosomal peptide synthetase), such as Bacillomycin D, Iturin A, Bacilosarcin B, Bacillcoumacin G and Macrolactin (N and G). These results suggest that the under-explored bacterial community of the Great Bitter Lake has a prospective biosynthetic capacity and can be a promising source for novel antibiotics.

Human hookworms from Argentina: Differential diagnosis of Necator americanus and Ancylostoma duodenale in endemic populations from Buenos Aires and Misiones

Hookworm infection is endemic in many countries throughout the world; however, the information about the prevalence of each species, Necatoramericanus and Ancylostomaduodenale, is inaccurate in many South American countries. We aimed to determine the prevalence of human hookworm species by combining the results of both microscopy and PCR among endemic populations in Argentina, represented by natives and immigrants. A total of 140 serial fecal specimens were obtained from natives in the province of Misiones and an immigrant community living in the province of Buenos Aires. Samples were examined using the formalin-ethyl acetate concentration technique (FECT) and one flotation technique (screening tests) and specific PCRs for N. americanus and A. duodenale. We characterized samples containing N. americanus by sequencing a fragment of the cytochrome b gene. The observed hookworm prevalence as assessed by the screening tests and PCR were 24.3% and 32.8%, respectively. PCR positive samples were identified as N. americanus. PCR had 100% sensitivity compared with 73.9% of screening tests. A total of 12 samples from individuals with hookworm-infected household members were positive only by PCR. N. americanus sequences showed 90.5% identity, being more similar to each other than to any of the sequences obtained from GenBank. This is the first study that provides molecular data and characterization of N. americanus in Argentina. The complementary use of FECT and one flotation technique to screen hookworm infections, followed by PCR to differentiate the species contribute to produce better prevalence estimates.

Characterisation of native <i>Bacillus thuringiensis</i> isolates toxicity to fall armyworm, <i>Spodoptera frugiperda</i> (J.E. Smith)

Toxicity of nine indigenous Bacillus thuringiensis (Bt) isolates collected from Tamil Nadu, India were tested against fall armyworm, Spodoptera frugiperda. At 30 ?g/ml concentration, two Bt isolates viz., T350 and T532 recorded 100 per cent mortality whereas isolates T527 and T532 registered 96 per cent mortality against neonate larvae of S. frugiperda in leaf dip bioassay. SDS PAGE analysis of spore crystal mixture revealed the presence of Cry1 and Cry2 proteins with visible bands at 130 kDa and 65 kDa. PCR screening results showed the presence of cry1 (cry1A, cry1Aa, cry1Ab), cry2 (cry2Aa, cry2Ab) in four isolates and vip3A genes in three isolates but cry9 gene was not present in any of the isolates tested.

Molecular characterization of extended-spectrum ß-lactamase producers, carbapenemase producers, polymyxin-resistant, and fosfomycin-resistant Enterobacterales among pigs from Egypt

ObjectivesTo perform the first prospective surveillance evaluating the occurrence of genes encoding colistin resistance, fosfomycin resistance, carbapenemase, or extended-spectrum ß-lactamases (ESBLs) among Enterobacterial isolates recovered from the gut flora of pigs from Egypt. MethodsBetween February and April 2020, 81 rectal swabs were collected from pigs in a slaughterhouse, Cairo, Egypt. Samples were screened for different resistance mechanisms using SuperPolymyxin, ChromID ESBL, SuperFOS, and SuperCarba selective agar plates. Antimicrobial susceptibility testing was performed for all isolates using disk diffusion and broth microdilution techniques. PCR screening was performed for ESBLs, carbapenemases, mcr, and fosA genes. Mating-out assays, multilocus sequence typing analysis, and plasmid typing were also performed. ResultsA high prevalence of ESBLs, carbapenemases, fosfomycin, and colistin resistance genes was evidenced among those isolates. The predominant ESBL identified was blaCTX-M-15, followed by blaCTX-M-9. We also identified blaNDM-5 and blaOXA-244. fosA3, fosA4, and fosA6 were identified in E. coli isolates. In addition, 11 MCR-1 producers were recovered. Notably, co-occurrence of ESBL genes and mcr or fosA genes was observed. MLST analysis revealed a high clonal diversity, ruling out the dissemination of one major clone. IncFIB-type was predominantly present among ESBL and FosA producers. The blaNDM-5 gene was carried on an IncX4-type, although the blaOXA-244 gene was chromosomally located. The mcr-1 gene was carried on a diversity of plasmids (IncI2, IncX4, and IncHI2). ConclusionThese results raise serious public health concerns as Egyptian pig meat could serve as a reservoir for antimicrobial resistance genes (ARGs), leading to worldwide dissemination.

Molecular detection of porcine parvovirus 1-associated reproductive failure in southern India.

This study used 56 aborted and stillborn fetuses from organized swine farms in Tamil Nadu and Kerala, southern states of India. All samples were screened by using a PCR assay that targets the NS1 gene for PPV. Furthermore, the PCR positive samples were subjected to amplification of the VP2 gene of PPV1 with designed primers and sequenced for further study. The PCR screening of 56 samples found that 14.3% (n = 8) were positive for PPV genome. According to VP2 gene–based PCR for PPV1, 897 bp specific amplicons were detected in all eight of the samples. Two of the eight positive samples (L17 and T5) were sequenced and annotated randomly. The BLAST analysis of contig sequence INDTNCHN-T5 revealed 100% sequence homology with Chinese PPV1genome, whereas sequence from INDTNCHN-L17 revealed 99.43% sequence homology with Spain, Chinese, and German. PPV1 sequences and both the sequences INDTNCHN-T5 and INDTNCHN-L17 were submitted to the GenBank under the accession numbers MW822566 and MW822567 respectively. A phylogenetic analysis of the sequences in this study revealed specific grouping along with PPV1 strains in cluster E. Amino acid analysis of both isolated sequences in addition to the reference sequence from PPV1 showed variations in position 215 (I to T) in both the isolates, variation at position 228 (Q to E) in T5 isolate and variations at position 59 (L to M) and 314 (K to E) in L17 isolate. This study represents the first report of PPV1 cluster E in Tamil Nadu, southern India.

Impact of Quorum Sensing System on Virulence Factors Production in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important pathogen that is frequently associated with nosocomial infections. The goal of this work was to determine the relationship between the quorum sensing system (QS) and the production of virulence factors in P. aeruginosa. A number of 100 P. aeruginosa isolates were collected from various clinical sources from different Mansoura university hospitals in the period from April 2018 till April 2019. PCR screening of QS genes in the isolates was carried out including lasI, lasR, rhlI and rhlR. Thereafter, assay of the production of different virulence factors in the isolates was established including biofilm formation, pyocyanin production, protease production, lipase production, hemolysin production as well as swimming motility. Finally, statistical analysis of the data was performed to confirm the relationship between the QS and the production of virulence factors. Out of the 100 P. aeruginosa isolates, 27 clinical isolates were QS deficient. PCR analysis revealed that 8 isolates lacked lasR gene, 15 isolates lacked lasR and rhlR genes, 1 isolate lacked lasR and lasI genes, 2 isolates lacked lasR, lasI and rhlR genes and 1 isolate lacked rhlR, rhlI and lasR genes. There was a significant decrease observed in the production of pyocyanin, protease, lipase, hemolysin and biofilm formation as well as swimming motility in P. aeruginosa QS deficient isolates in comparison to non-QS deficient ones. There was a clear association between QS and virulence factors production in P. aeruginosa. This could open the door for novel promising targets for developing new therapeutic strategies against infections caused by this pathogen.

Glycosylation of Semi-Synthetic Isoflavene Phenoxodiol with a Recombinant Glycosyltransferase from Micromonospora echinospora ATCC 27932.

Glycosyltransferase (GT)-specific degenerate PCR screening followed by in silico sequence analyses of the target clone was used to isolate a member of family1 GT-encoding genes from the established fosmid libraries of soil actinomycetes Micromonospora echinospora ATCC 27932. A recombinant MeUGT1 was heterologously expressed as a His-tagged protein in E. coli, and its enzymatic reaction with semi-synthetic phenoxodiol isoflavene (as a glycosyl acceptor) and uridine diphosphate-glucose (as a glycosyl donor) created two different glycol-attached products, thus revealing that MeUGT1 functions as an isoflavonoid glycosyltransferase with regional flexibility. Chromatographic separation of product glycosides followed by the instrumental analyses, clearly confirmed these previously unprecedented glycosides as phenoxodiol-4'-α-O-glucoside and phenoxodiol-7-α-O-glucoside, respectively. The antioxidant activities of the above glycosides are almost the same as that of parental phenoxodiol, whereas their anti-proliferative activities are all superior to that of cisplatin (the most common platinum chemotherapy drug) against two human carcinoma cells, ovarian SKOV-3 and prostate DU-145. In addition, they are more water-soluble than their parental aglycone, as well as remaining intractable to the simulated in vitro digestion test, hence demonstrating the pharmacological potential for the enhanced bio-accessibility of phenoxodiol glycosides. This is the first report on the microbial enzymatic biosynthesis of phenoxodiol glucosides.

Brucella spp. distribution, hosting ruminants from Greece, applying various molecular identification techniques

BackgroundBrucellosis still remains an endemic disease for both livestock and human in Greece, influencing the primary sector and national economy in general. Although farm animals and particularly ruminants constitute the natural hosts of the disease, transmission to humans is not uncommon, thus representing a serious occupational disease as well. Under this prism, knowledge concerning Brucella species distribution in ruminants is considered a high priority. There are various molecular methodologies for Brucella detection with however differential discriminant capacity. Hence, the aim of this survey was to achieve nationally Brucella epidemiology baseline genotyping data at species and subtype level, as well as to evaluate the pros and cons of different molecular techniques utilized for detection of Brucella species. Thirty-nine tissue samples from 30 domestic ruminants, which were found positive applying a screening PCR, were tested by four different molecular techniques i.e. sequencing of the 16S rRNA, the BP26 and the OMP31 regions, and the MLVA typing panel 1 assay of minisatellite markers.ResultsOnly one haplotype was revealed from the 16S rRNA sequencing analysis, indicating that molecular identification of Brucella bacteria based on this marker might be feasible solely up to genus level. BP26 sequencing analysis and MLVA were in complete agreement detecting both B. melitensis and B. abortus. An interesting exception was observed in 11 samples, of lower quality extracted DNA, in which not all expected MLVA amplicons were produced and identification was based on the remaining ones as well as on BP26. On the contrary OMP31 failed to provide a clear band in any of the examined samples.ConclusionsThe present study reveals the constant circulation of Brucella bacteria in ruminants throughout Greece. Further, according to our results, BP26 gene represents a very good alternative to MLVA minisatellite assay, particularly in lower quality DNA samples.

Development of Superior Fibre Quality Upland Cotton Cultivar Series 'Ravnaq' Using Marker-Assisted Selection.

Marker-assisted selection (MAS) helps to shorten breeding time as well as reduce breeding resources and efforts. In our MAS program, we have targeted one of previously reported LD-blocks with its simple sequence repeat (SSR) marker(s), putatively associated with, at least, four different fibre quality QTLs such as fibre length, strength, micronaire and uniformity. In order to transfer targeted QTLs from a donor genotype to a cultivar of choice, we selected G. hirsutum donor genotypes L-141 and LN-1, possessing a fibre quality trait-associated LD-block from the chromosome 7/16. We crossed the donor lines with local elite G. hirsutum cultivars ‘Andijan-35’ and ‘Mekhnat’ as recipients. As a result, two segregating populations on LD-block of interest containing fibre QTLs were developed through backcrossing (BC) of F1 hybrids with their relative recipients (used as recurrent parents) up to five generations. In each BC and segregating BC1-5F1 populations, a transfer of targeted LD-block/QTLs was monitored using a highly polymorphic SSR marker, BNL1604 genotype. The homozygous cultivar genotypes with superior fibre quality and agronomic traits, bearing a targeted LD-block of interest, were individually selected from self-pollinated BC5F1 (BC5F2–5) population plants using the early-season PCR screening analysis of BNL1604 marker locus and the end-of-season fibre quality parameters. Only improved hybrids with superior fibre quality compared to original recipient parent were used for the next cycle of breeding. We successfully developed two novel MAS-derived cotton cultivars (named as ‘Ravnaq-1’ and ‘Ravnaq-2’) of BC5F5 generations. Both novel MAS cultivars possessed stronger and longer fibre as well as improved fibre uniformity and micronaire compared to the original recurrent parents, ‘Andijan-35’ and ‘Mekhnat’. Our efforts demonstrated a precise transfer of the same LD-block with, at least, four superior fibre QTLs in the two independent MAS breeding experiments exploiting different parental genotypes. Results exemplify the feasibility of MAS in cotton breeding.

Antimicrobial resistance among Haemophilus influenzae isolates responsible for lower respiratory tract infections in Poland, 2005-2019.

Haemophilus influenzae is a human-specific pathogen responsible for respiratory tract infections, meningitis, and sepsis. The study aimed to characterize antibiotic resistance in H. influenzae strains isolated from patients with lower respiratory tract infections over 15years in Poland. The minimuminhibitory concentrations (MICs) of clinically relevant antibiotics were determined by broth microdilution method. Screening for beta-lactam resistance was performed in all isolates following EUCAST recommendation. Finally, relevant changes in penicillin-binding protein 3 (PBP3) were detected by PCR screening. Of the 1481 isolates collected between 2005 and 2019, 12.6%, 0.2%, 17.1%, and 0.2% were resistant to ampicillin, amoxicillin/clavulanate, cefuroxime, and ceftriaxone, respectively. Among them, 74.4% (1102/1481) of isolates were categorized as BLNAS (β-lactamase negative, ampicillin-susceptible), 13.0% (192/1481) as BLNAS with modified PBP3 (mutations in ftsI gene), 2.6% (39/1481) as BLNAR (β-lactamase negative, ampicillin-resistant), and 0.2% had PBP3 modifications typical for high-BLNAR. Production of β-lactamase characterized 9.7% of isolates (8.6% BLPAR-β-lactamase-positive, ampicillin-resistant, and 1.1% BLPACR-β-lactamase-positive, amoxicillin-clavulanate resistant). Three isolates with PBP3 modifications typical for high-BLNAR proved resistant to ceftriaxone (MIC > 0.125mg/L). Resistance to ciprofloxacin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole was observed in 0.1%, 0.5%, 1.6%, and 24.7% of isolates, respectively. This is the first report of Polish H. influenzae isolates resistant to third-generation cephalosporins. Polish H. influenzae isolates demonstrate similar susceptibility trends as in many other countries. The substantial proportion of β-lactam-resistant isolates and the emergence of those resistant to third-generation cephalosporins are of great concern and should be under surveillance.

Characterization of indigenous Bacillus thuringiensis isolate RM11 toxic to the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae)

BackgroundBacillus thuringiensis (Bt) Berliner is an omnipresent soil bacterium used as world’s leading biopesticide to combat agriculturally important insect pests. This study was aimed at protein and gene profiling of an indigenous Bt isolate RM11, which was toxic to the larvae of diamondback moth, Plutella xylostella (L.) in laboratory bioassays.ResultsIndigenous Bt isolate RM11 was characterized along with the standard checks B. thuringiensis subsp. kurstaki (Btk) HD1 and 78/11, based on colony characters, protein profile and PCR screening. All three Bt colonies were fried egg type, white in color with flat elevation and undulated margin. PCR screening revealed the presence of cry1Ac and vip3A genes, which encode lepidopteran toxic proteins in RM11. SDS-PAGE results showed the presence of a prominent protein band of cry1Ac, vip3A with molecular weights 135 kDa, 88 kDa and other bands at 70, 50, 32 and 10 kDa. In leaf disk bioassay with spore crystal mixture, RM11 exhibited toxicity with LC50 of 4.51 µg/ml as against 0.07 µg/ml in positive standard HD1, based on mortality at 72 h after treatment. At LC50 of 4.51 µg/ml, solubilized and insolubilized protein of RM11 was found to produce 56 and 70% mortality.ConclusionsThe present study revealed that RM11 could be a viable alternative for consideration in developing a native Bt formulation and for inclusion in the integrated management of P. xylostella with other native isolates producing different toxins. Furthermore, these findings imply that RM11 could be a source of new cry toxin, which can be confirmed through whole-genome sequencing analysis.

Exposure of Primate Reservoir Hosts to Mosquito Vectors in Malaysian Borneo

Several vector-borne pathogens of primates have potential for human spillover. An example is the simian malaria Plasmodium knowlesi which is now a major public health problem in Malaysia. Characterization of exposure to mosquito vectors is essential for assessment of the force of infection within wild simian populations, however few methods exist to do so. Here we demonstrate the use of thermal imaging and mosquito magnet independence traps (MMIT) to assess the abundance, diversity and infection rates in mosquitoes host seeking near long-tailed macaque (Macaca fasicularis) sleeping sites in the Lower Kinabatangan Wildlife Sanctuary, Malaysian Borneo. The primary Plasmodium knowlesi vector, Anopheles balabacensis, was trapped at higher abundance near sleeping sites than control trees. Although none of the An. balabacensis collected (n = 15) were positive for P. knowlesi by PCR screening, two were infected with another simian malaria Plasmodium inui. Analysis of macaque stools from sleeping sites confirmed a high prevalence of Plasmodium infection, suspected to be P. inui. Recently, natural transmission of P. inui has been detected in humans and An. cracens in Peninsular Malaysia. The presence of P. inui in An. balabacensis here and previously in human-biting collections highlight its potential for spillover from macaques to humans in Sabah. We advocate the use of MMITs for non-invasive sampling of mosquito vectors that host seek on wild simian populations.

SARS-CoV-2 RT-qPCR testing of pooled saliva samples: A case study of 824 asymptomatic individuals and a questionnaire survey in Japan.

From the beginning of the COVID-19 pandemic, the demand for diagnostic and screening tests has exceeded supply. Although the proportion of vaccinated people has increased in wealthier countries, breakthrough infections have occurred amid the emergence of new variants. Pooled-sample COVID-19 testing using saliva has been proposed as an efficient, inexpensive, and non-invasive method to allow larger-scale testing, especially in a screening setting. In this study, we aimed to evaluate pooled RT-qPCR saliva testing and to compare the results with individual tests. Employees of Philips Japan, Ltd. were recruited to participate in COVID-19 screening from October to December 2020. Asymptomatic individuals (n = 824) submitted self-collected saliva samples. Samples were tested for the presence of SARS-CoV-2 by RT-qPCR in both 10-sample pools and individual tests. We also surveyed participants regarding their thoughts and behaviors after the PCR screening project. Two of the 824 individuals were positive by RT-qPCR. In the pooled testing, one of these two had no measurable Ct value, but showed an amplification trend at the end of the PCR cycle. Both positive individuals developed cold-like symptoms, but neither required hospitalization. Of the 824 participants, 471 responded to our online questionnaire. Overall, while respondents agreed that PCR screening should be performed regularly, the majority were willing to undergo PCR testing only when it was provided for free or at low cost. In conclusion, pooled testing of saliva samples can support frequent large-scale screening that is rapid, efficient, and inexpensive.

Natural Compounds as a Promising Therapeutic Agent for SPOP Downregulated Breast Cancer

Breast cancer is the second most deadly type of cancer for women in America. Due to this, there is a pressing need to investigate and develop a better treatment plan for those afflicted by it. There are numerous studies that show a downregulation of the E3 ubiquitin ligase Type POZ Protein (SPOP) for up to 70% of breast cancer patients. This demonstrates that, in breast cancer, SPOP is a vital tumor suppressor gene. In previous research projects, our lab has shown that SPOP targets GLI3 for ubiquitination and degradation which subsequently places a restraint on SHH signaling. Furtermore, we have shown that SPOP mutations lead to a upregulation of GLI3 and subsequent hyperactivated SHH signaling in prostate cancer. Since SPOP is downregulated in a large number of breast cancer patients, we hypothesize that SPOP downregulated breast cancer tumors would also display hyperactivated SHH signaling. Additionally, we hypothesize that SPOP downregulated breast cancer patients will benefit from therapeutics that specifically target the SHH signaling pathway. To characterize the effect of SPOP downregulation in breast cancer, we used a lentivirus with shRNA complementary to SPOP to generate a stable MCF‐7 SPOP knockdown cell line. Proliferation assays were utilized to first confirm that SPOP knockdown promotes enhanced cell proliferation. Next, quantitative PCR was used to demonstrate that SPOP knockdown leads to an upregulation of GLI3 target genes. To find a novel treatment strategy for SPOP downregulated breast cancer, a natural compound library was used. Through the screen and subsequent quantitative PCR analysis, we identified novel therapeutic natural compounds that specifically target SPOP downregulated MCF‐7 cells in a manner that involves disruption of GLI3‐dependent SHH signaling. Our findings thus give necessary insight into novel treatment strategies for patients suffering from SPOP downregulated breast cancer.

Molecular detection of endosymbionts in local populations of Helicoverpa armigera (Lepidoptera: Noctuidae) in European part of Russia

Cotton bollworm Helicoverpa armigera is one of the most polyphagous and cosmopolitan pests. Intracellular endosymbionts are widespread in Lepidoptera, often playing an important role in their dynamics. The prevalence of endosymbionts of cotton bollworm in Russia was not investigated. Cotton bollworm larvae and adults were collected in 2018–2020 in Krasnodar Area, and in Voronezh and Saratov Regions (from 131 to 170 insects) and analyzed by PCR using sets of group-specific primers for baculoviruses (locus lef8), bacteria of the genus of Wolbachia (locus wsp), and microsporidia (locus SSU rRNA). Level of infection with baculoviruses was 16 % for the sample of 32 individuals collected in Temryuk District of Krasnodar Area in 2018. The infection rate of the entire sample of 170 individuals was 2.9 %. The lef8 locus demonstrated 98.7–99.6 % of sequence similarity to the nuclear polyhedrosis virus isolates from the cotton bollworm and American bollworm. Among the tested 131 insects, bacteria of the genus of Wolbachia were not detected. PCR screening for microsporidia revealed one positive larvae among 19 insects collected in Krasnoarmeysk District of Krasnodar Area in 2019, which corresponded to the prevalence of 5 %. Partial sequencing of the genes coding for SSU rRNA and largest subunit RNA polymerase II made it possible to identify the new isolate as N. bombycis.

REPEATED SAMPLING OF WILD INDIVIDUALS REVEALS OPHIDIOMYCES OPHIDIICOLA INFECTION DYNAMICS IN A PENNSYLVANIA SNAKE ASSEMBLAGE.

Ophidiomyces ophidiicola is an emerging fungal pathogen associated with infections in snakes across North America. Although documented in Pennsylvania, O. ophidiicola has not been found at Powdermill Nature Reserve (PNR) in southwestern Pennsylvania, where the snake assemblage has been studied since 2002 and several species have recently declined. We surveyed for O. ophidiicola and putative ophidiomycosis at PNR. We screened five species of free-ranging, wild snakes (n=34) for suspected ophidiomycosis by visually checking for dermatitis and swabbing for the presence of O. ophidiicola DNA. We found a moderate prevalence of snakes with skin lesions (n=15) but a low prevalence of snakes with O. ophidiicola DNA in traditional PCR assays (n=2). Both positive snakes belonged to the same species and only one presented with lesions. When quantitative PCR screens were performed on duplicate swabs, 19 snakes were positive for O. ophidiicola DNA, with positive individuals in two species. Mark-recapture methods revealed seasonal variability in disease dynamics for sampled snakes. One individual presented with less than five skin lesions and tested negative in May 2020, had more than five lesions with a high fungal DNA load in June 2020, and no lesions with a low fungal DNA load in July 2020. We also found that snakes sampled from under the same cover object at the same time either all tested positive or all negative, including one instance involving two species. Our results underscore the value of using multiple screening techniques for O. ophidiicola surveillance and repeated sampling of individuals to understand the dynamics of ophidiomycosis in wild populations as compared to single method and single timepoint approaches.

DETECTION OF SPLENDIDOFILARIA SP. (ONCHOCERCIDAE:SPLENDIDOFILARIINAE) MICROFILARIA WITHIN ALASKAN GROUND-DWELLING BIRDS IN THE GROUSE SUBFAMILY TETRAONINAE USING TAQMAN PROBE-BASED REAL-TIME PCR.

Grouse and ptarmigan (Galliformes) harbor fairly diverse helminth faunas that can impact the host's health, including filarial nematodes in the genus Splendidofilaria. As host and parasite distributions are predicted to shift in response to recent climate change, novel parasites may be introduced into a region and impose additional stressors on bird populations. Limited information is available on the prevalence of filariasis in Alaska galliforms. To date, no molecular surveys have been completed. Past studies relied on examining blood smears or total body necropsies, which are time-consuming and may not detect filarial parasites with low prevalence in hosts. Therefore, we developed a TaqMan probe-based real-time PCR assay targeting the cytochrome c oxidase 1 gene (COI) of Splendidofilaria to decrease processing times and increase sensitivity as well as provide baseline data on the diversity of filariid infections in galliform species in Alaska. We screened a combined total of 708 galliform samples (678 unique individual birds) from different tissues (blood, muscle, and lung) for the presence of filarial DNA across the state of Alaska. Real-time PCR screening revealed an overall prevalence of filarial infection of 9.5% across species: Bonasa umbellus (0%, n = 23), Dendragapus fuliginosus (0%, n = 8), Falcipennis canadensis (26.8%, n = 198), Lagopus lagopus (2.6%, n = 274), Lagopus leucura (0%, n = 23), Lagopus muta (3%, n = 166), and Tympanuchus phasianellus (12.5%, n = 16). We observed microfilarial infections throughout most of Alaska except in Arctic regions and the Aleutian Islands where viable vectors may not be present.

Copy number assessment of SMN1 based on real-time PCR with high-resolution melting: fast and highly reliable testing

BackgroundSpinal muscular atrophy (SMA) is a neuromuscular disease mainly caused by the absence of both copies of the survival motor neuron 1 (SMN1) gene. Multiple regions recommended population-wide SMA screening to quantify the copy number of SMN1. SMN1 diagnostic assays for the simplified procedure, high sensitivity, and throughput continue to be needed. MethodsReal-time PCR with high-resolution melting for the quantifying of the SMN1 gene exon 7 copies and exon 8 copies were established and confirmed by multiplex ligation-dependent probe amplification (MLPA). The diagnosis of 2563 individuals, including SMA patients, suspected cases, and the general population, was tested by real-time PCR. The results were compared with the gold standard test MLPA. ResultsIn this study, the homozygous and heterozygous deletions were detected by real-time PCR with a high-resolution melting method with an incidence of 10.18% and 2.26%, respectively. In addition, the R-value distribution (P > 0.05) among 8 replicates and the coefficient of variation (CV < 0.003) suggested that the real-time PCR screening test had high reproducibility. High concordance was obtained between real-time PCR with high-resolution melting and MLPA. ConclusionsThe real-time PCR based on high-resolution melting provides a sensitive and high-throughput approach to large-scale SMA carrier screening with low cost and labor.

Clinical characterization of COVID-19 breakthrough infections, Philippines.

We clinically characterized PCR detected breakthrough infections among partially/fully vaccinated cases with majority given an inactivated vaccine, CoronaVac. From 1 March to 15 July 2021, we detected 182 SARS-CoV-2 infections among vaccinated cases with 129 classified as breakthrough infections. Majority were male, 30–39 y.o., and were asymptomatic or mildly symptomatic with few severe cases. Alpha, Beta and Delta VOCs were detected from sequenced breakthrough infections. Healthcare workers had significantly lower Ct values(higher viral loads) versus non-HCWs. Our results underscore the importance of regular PCR screening for HCWs due to the risk of SARS-CoV-2 transmission from asymptomatic breakthrough infections and provide evidence supporting administration of a booster dose especially to HCWs

CRISPR-Cas9-mediated chromosome engineering in Arabidopsis thaliana.

The rise of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system has made it possible to induce double-strand breaks at almost any desired target site in the genome. In plant somatic cells, double-strand breaks are predominantly repaired by the error-prone nonhomologous end-joining pathway, which can lead to mutations at the break site upon repair. So far, it had only been possible to induce genomic changes of up to a few hundred kilobases in plants utilizing this mechanism. However, by combining the highly efficient Staphylococcus aureus Cas9 (SaCas9) with an egg-cell-specific promoter to facilitate heritable mutations, chromosomal rearrangements in the Mb range, such as inversion and translocations, were obtained in Arabidopsis thaliana recently. Here we describe the chromosome-engineering protocol used to generate these heritable chromosomal rearrangements in A. thaliana. The protocol is based on Agrobacterium-mediated transformation of A. thaliana with transfer DNA constructs containing SaCas9, which is driven by an egg-cell-specific promoter, and two guide RNAs that have been preselected based on their cutting efficiency. In the T1 generation, primary transformants are selected and, if required, analyzed by Droplet Digital PCR and propagated. In the following generations, junction-specific PCR screenings are carried out until plants that carry the rearrangement homozygously are identified. Using this protocol, overall rearrangement frequencies range between 0.03% and 0.5%, depending on the type of rearrangement. In total, it takes about 1 year to establish homozygous lines.