Detection and Quantification of Stagonosporopsis cucurbitacearum in Seeds of Cucurbita maxima Using Droplet Digital Polymerase Chain Reaction

Stagonosporopsis cucurbitacearum is an important seedborne pathogen of squash (Cucurbita maxima). The aim of our work was to develop a rapid and sensitive diagnostic tool for detection and quantification of S. cucurbitacearum in squash seed samples, to be compared with blotter analysis, that is the current official seed test. In blotter analysis, 29 of 31 seed samples were identified as infected, with contamination from 1.5 to 65.4%. A new set of primers (DB1F/R) was validated in silico and in conventional, quantitative real-time PCR (qPCR) and droplet digital (dd) PCR. The limit of detection of S. cucurbitacearum DNA for conventional PCR was ∼1.82 × 10–2 ng, with 17 of 19 seed samples positive. The limit of detection for ddPCR was 3.6 × 10–3 ng, which corresponded to 0.2 copies/μl. Detection carried out with artificial samples revealed no interference in the absolute quantification when the seed samples were diluted to 20 ng. All seed samples that showed S. cucurbitacearum contamination in the blotter analysis were highly correlated with the absolute quantification of S. cucurbitacearum DNA (copies/μl) in ddPCR (R2 = 0.986; p ≤ 0.01). Our ddPCR protocol provided rapid detection and absolute quantification of S. cucurbitacearum, offering a useful support to the standard procedure.

Comparative Evaluation of Diagnostic Performance of Circulating free DNA by Conventional vs. Real Time PCR in Gallbladder Cancer

Background: Circulating free DNA (cfDNA) in serum/plasma has been studied as a promising biomarker in various pathologies, including cancer. However, there is no standardized method for the isolation and quantification of serum cfDNA. An effective and reliable method for isolation and quantification is of utmost importance before any clinical decision. The current study compares the conventional and real-time PCR methods to find any differences and concordance in cfDNA levels between the two methods and the diagnostic accuracy of cfDNA by each method. Methods: Serum sample was collected from 67 subjects, including 17 normal healthy individuals (control, n=17), 19 disease controls (cholecystitis, n=19), and 31 Gallbladder cancer patients (cancer, n=31) before any treatment for cfDNA quantification. Results: The cfDNA level did not differ significantly between two methods in both control and cholecystitis groups. In cancer group, cfDNA level was significantly (P < 0.001) different and higher in real time PCR as compared to conventional PCR. There was no significant correlation between two methods in control (r=0.02, P = 0.937), cholecystitis (r=0.10, P = 0.697), cancer (r=-0.08, P = 0.657) and total cases (control + cholecystitis + cancer) (r=0.06, P = 0.622). The diagnostic accuracy of two methods was found similar (P > 0.05) when assessed between control vs. cholecystitis (Z=0.85, P = 0.397), and control vs. total cases (Z=1.35, P = 0.177). However, the diagnostic accuracy of real time PCR was found significantly different and higher as compared to conventional PCR when assessed between control vs. cancer (Z=2.98, P = 0.003), and cholecystitis vs. cancer (Z=4.41, P < 0.001). Conclusion: Quantitative real-time PCR method is of high accuracy, reproducibility, and time-effectiveness. The diagnostic accuracy of real-time PCR was higher compared to conventional PCR.

COVIDFast: A high-throughput and RNA extraction-free method for SARS-CoV-2 detection in swab (SwabFAST) or saliva (SalivaFAST)

There is an urgent need of having a rapid, high throughput, yet accurate SARS-COV-2 PCR testing to control the COVID19 pandemic. However, the RNA extraction step in conventional PCR creates a major bottle neck in the diagnostic process. In this paper we modified the CDC COVID-19 assay and developed an RNA-extraction free RT-qPCR assay for SARS-CoV-2, i.e. COVIDFast. Depending on sample types, the assay is further divided into SwabFAST, which uses anterior nares nasal swab, and SalivaFAST, which uses saliva. By utilizing the proprietary buffer for either swab or saliva samples, the performance of SwabFAST or SalivaFAST is equivalent to RNA-extraction SARS-CoV-2 RT-qPCR in both contrived and clinical samples. The limit of detection of either assay is 4 copies/uL. We further developed a semi-automatic system, which is easy to adapt by clinical lab for implementation of a high-throughput SARS-CoV-2 test. Working together with the COVIDCheck Colorado, we have tested over 400,000 samples using COVIDFast (83.62% SwabFAST and 16.38% SalivaFAST) in less than a year, resulting in significant clinical contribution in the battle against COVID-19 during the pandemic.

Isolation and Molecular Detection of Mycoplasma gallisepticum in Commercial Layer Chickens in Sylhet, Bangladesh

Mycoplasma gallisepticum induced poultry diseases are associated with a huge economic crisis and have a considerable impact on the poultry industry worldwide. The aim of the current study was to isolate and perform molecular detection of MG circulating pathogenic strain in the commercial layer farms in the Sylhet district of Bangladesh. The entire study was conducted from January 2018 to January 2019 at three Upazilas of Sylhet district in Bangladesh. A total of 50 dead layer chickens (indicating signs of respiratory distress before death) were collected randomly from 15 different layer farms. The tissue samples, such as air sacs, trachea, and lungs, were taken from suspected dead chickens. Both cultural and PCR-based techniques were applied to identify Mycoplasma from tissue samples. The conventional PCR technique was implemented to amplify 185 bp DNA fragments for the MG. Out of 50 samples, 36% (18/50) and 70% (35/50) of MG were identified by cultural method and PCR, respectively. Based on the results of the study, it can be concluded that PCR is an easier, more sensitive, and less time-consuming method for the early diagnosis of MG in chickens, compared to cultural isolation and hence can lower the economic burden to poultry farmers caused by this disease.

A long-amplicon viability-qPCR test for quantifying living pathogens that cause bacterial spot in tomato seed

Bacterial spot is one of the most serious diseases of tomato. It is caused by four species of Xanthomonas: X. euvesicatoria, X. gardneri, X. perforans, and X. vesicatoria. Contaminated and/or infected seed can serve as a major source of inoculum for this disease. The use of certified pathogen-free seed is one of the primary management practices to reduce the inoculum load in commercial production. Current seed testing protocols rely mainly on plating the seed extract and conventional PCR, however, the plating method cannot detect viable but non-culturable cells and the conventional PCR assay has limited capability to differentiate DNA extracted from viable versus dead bacterial cells. To improve the sensitivity and specificity of the tomato seed testing method for the bacterial spot pathogens, a long-amplicon qPCR assay coupled with propidium monoazide (PMA-qPCR) was developed to quantify selectively the four pathogenic Xanthomonas species in tomato seed. The optimized PMA-qPCR procedure was evaluated on pure bacterial suspensions, bacteria-spiked seed extracts, and seed extracts of inoculated and naturally-infected seed. A crude DNA extraction protocol also was developed and PMA-qPCR with crude bacterial DNA extracts resulted in accurate quantification of 104-108 CFU/ml of viable bacteria when mixed with dead cells at concentrations as high as 107 CFU/ml in the seed extracts. With DNA purified from concentrated seed extracts, the PMA-qPCR assay was able to detect DNA of the target pathogens in seed samples spiked with ≥75 CFU/ml (~0.5 CFU/seed) of the viable pathogens. Latent class analysis of the inoculated and naturally-infected seed samples showed that the PMA-qPCR assay had greater sensitivity than plating the seed extracts on the semi-selective MTMB and CKTM media for all four target species. Being much faster and more sensitive than dilution plating, the PMA-qPCR assay has a promising potential to serve as a standalone tool or used in combination with the plating method to improve tomato seed testing and advance the production of clean seed.

Rapid detection of Enterococcus and vancomycin resistance using recombinase polymerase amplification

Vancomycin-resistant enterococci (VRE), especially Enterococcus faecium, have been a global concern, often causing serious healthcare-associated infections. We established a rapid approach for detecting E. faecium and vancomycin-resistance genes (vanA and vanB) in clinical samples using isothermal recombinase polymerase amplification (RPA) combined with a lateral-flow (LF) strip. Specific RPA primer sets and probes for ddl (to identify the presence of E. faecium) vanA and vanB genes were designed. The RPA reaction was performed under isothermal condition at 37 °C within 20 min and read using the LF strip within a further 5 min. A total of 141 positive blood-cultures and 136 stool/rectal swab samples were tested using RPA-LF method compared to the conventional PCR method. The RPA-LF method exhibited 100% sensitivity in both blood-culture (60 E. faecium; 35 vanA type and two vanB type) and stool/rectal-swab samples (63 E. faecium and 36 vanA type) without cross-reaction (100% specificity). The lower detection limit of the RPA-LF was approximately 10 times better than that of the conventional PCR method. The RPA-LF method is an alternative rapid method with excellent sensitivity and specificity for detecting E. faecium, vanA, and vanB, and it has the potential to be used as a point-of-care device for VRE therapy and prevention.

Rapid detection of a novel B1-β-lactamase gene, blaAFM-1 using a loop-mediated isothermal amplification (LAMP) assay

Background BlaAFM-1 (GenBank Accession No. 143105.1) is a new B1 subclass metallo-β-lactamase gene discovered by our group, and isolated from an Alcaligenes faecalis plasmid that renders carbapenem antibiotics ineffective. In this study, we generated a fast and reliable assay for blaAFM-1 detection. Methods We designed optimum loop-mediated isothermal amplification (LAMP) primers and constructed a recombinant plasmid AFM-1 to specifically detect blaAFM-1. Optimal LAMP primers were used to assess sensitivity of the recombinant plasmid AFM-1 and blaAFM-1-supplemented samples (simulated sputum and simulated feces). Fifty two samples, without blaAFM-1, were used to assess LAMP real-time assay specificity; these samples were verified by conventional PCR and sequencing for the absence of blaAFM-1. Three hundred clinical Gram-negative carbapenem-resistant strains were tested by LAMP assay for strains carrying blaAFM-1, which were confirmed by conventional PCR and Sanger sequencing. We calculated the sensitivity and its 95% confidence interval (95% CI), specificity and its 95% CI, and predictive values of the LAMP assay and conventional PCR/sequencing by investigating positive and negative clinical strains. Results The lowest limit of detection for the recombinant plasmid AFM-1 and blaAFM-1-supplemented samples (in both simulated sputum and simulated feces) was 101 copies/reaction. All amplification curves of the 52 blaAFM-1-free bacteria strains were negative, suggesting the LAMP assay had excellent specificity for detecting blaAFM-1. Among the 300 clinical strains, eight were positive for blaAFM-1 using LAMP. These LAMP results were consistent with conventional PCR and Sanger sequencing data. As with conventional PCR/sequencing, the LAMP method exhibits 100% sensitivity (95% CI 59.8–100%) and 100% specificity (95% CI 98.4–100%) for blaAFM-1 detection. The LAMP assay is also time-efficient (1 h) for blaAFM-1 detection. Conclusions We established a new LAMP assay with high sensitivity and specificity to detect the novel B1-β-lactamase gene, blaAFM-1.

Viral metagenomics reveals two novel anelloviruses in feces of experimental rats

Background Rodents are widely distributed and are the natural reservoirs of a diverse group of zoonotic viruses. Thus, analyzing the viral diversity harbored by rodents could assist efforts to predict and reduce the risk of future emergence of zoonotic viral diseases. Rodents are commonly used in animal testing, particularly mice and rats. Experimental rats are important animal models, and a history of pathogenic infections in these animals will directly affect the animal trial results. The pathogenicity of Anellovirus (AV) remains poorly understood due to the lack of a suitable model cell line or animal to support the viral cycle. This study aimed to discover possible anelloviruses from the virome in feces of experimental rats by viral metagenomic technique. Methods Fecal samples were collected from 10 commercial SD rats and pooled into a sample pool and then subjected to libraries construction which was then sequenced on Illumina MiSeq platform. The sequenced reads were analyzed using viral metagenomic analysis pipeline and two novel anelloviruses (AVs) were identified from fecal sample of experimental rats. The prevalence of these two viruses was investigated by conventional PCR. Results The complete genomic sequence of these two AVs were determined and fully characterized, with strain name ratane153-zj1 and ratane153-zj2. The circular genomes of ratane153-zj1 and ratane153-zj2 are 2785 nt and 1930 nt in length, respectively, and both include three ORFs. Ratane153-zj1 closely clustered with members within the genus Wawtorquevirus and formed a separate branch based on the phylogenetic tree constructed over the amino acid sequence of ORF1 of the two AVs identified in this study and other related AVs. While the complete amino acid sequences of ORF1 of ratane153-zj2 (nt 335 to 1390) had the highest sequence identity with an unclassified AV (GenBank No. ATY37438) from Chinchilla lanigera, and they clustered with one AV (GenBank No. QYD02305) belonging to the genus Etatorquevirus from Lynx rufus. Conventional PCR with two sets of specific primers designed based on the two genomes, respectively, showed that they were detectable at a low frequency in cohorts of experimental rats. Conclusion Our study expanded the genome diversity of AVs and provided genetic background information of viruses existed in experimental rats.

Emergence of a novel recombinant of CV-A5 in HFMD epidemics in Xiangyang, China

Background Hand, foot and mouth disease (HFMD) is caused by a variety of enterovirus serotypes and the etiological spectrum worldwide has changed since a large scale of outbreaks occurred in 1997. Methods A large number of clinical specimens of HFMD patients were collected in Xiangyang and genotyping was performed by qRT-PCR, conventional PCR amplification and sequencing. Among the 146 CV-A5 detected cases, the complete genome sequences of representative strains were determined for genotyping and for recombination analysis. Results It was found that CV-A5 was one of the six major serotypes that caused the epidemic from October 2016 to December 2017. Phylogenetic analyses based on the VP1 sequences showed that these CV-A5 belonged to the genotype D which dominantly circulated in China. Recombination occurred between the CV-A5 and CV-A2 strains with a breakpoint in the 2A region at the nucleotide 3791. Conclusions The result may explain the emergence of CV-A5 as one of the major pathogens of HFMD. A multivalent vaccine against HFMD is urgently needed to control the disease and to prevent emerging and spreading of new recombinants.