Specific and Rapid Detection of Lily symptomless virus and Arabis mosaic virus in Lily by Dual IC‐RT‐PCR

Lily symptomless virus (LSV) and Arabis mosaic virus (ArMV) cause severe losses of quantity and quality of lily flower and bulb production. Specificity, sensitivity and speed of detection methods for viruses need to be improved greatly to prevent LSV and ArMV from spreading from infected lilies. A dual IC‐RT‐PCR procedure for detection was developed in which the antibodies of LSV and ArMV were mixed and the mixture used to coat the PCR tubes. The particles of the two viruses were captured by the respective antibodies. Interference by other RNA viruses in infected lily was eliminated in the RT‐PCR. Also, an RNA extraction step was omitted. The dual IC‐RT‐PCR products of LSV and ArMV were 521 bp and 691 bp, respectively. The specificity of the method was validated; only LSV and ArMV of four viruses were detected by dual IC‐RT‐PCR. The sensitivity of the detection method is 1 mg leaf tissue and higher than DAS‐ELISA due to enrichment by dual immunocapture.     

A Cocktail ELISA Semi‐nested RT‐PCR Assay to Detect Bean pod mottle virus in Soya bean Seeds

Bean pod mottle virus (BPMV) has been identified as an important pathogen for plant quarantine in China because large quantities of soya bean seeds (approximately 7 × 10⁷ tons) are imported annually. To develop a practical detection programme for BPMV, a cocktail enzyme‐linked immunosorbent assay (ELISA) nested RT‐PCR using a combination of serological and molecular methods was designed for soya bean seeds. The single‐vessel detection assay was performed in a 96‐well ELISA plate, which served as a carrier for the subsequent nested RT‐PCR assay. Assay specificity was demonstrated by the production of the expected 330‐ and 296‐bp bands using the external and internal primers, respectively. This method was 10⁴‐fold more sensitive than immunocapture‐RT‐PCR (IC‐RT‐PCR). In particular, it is important to note that this assay resulted in successful micro‐extraction from soya bean seeds and combined the advantages of each individual technique. The cocktail ELISA nested RT‐PCR is a specific, sensitive, rapid and economical procedure to rapidly identify and characterize BPMV and could be suitable for both primary‐level platforms and laboratories.     

Seroprevalence of Helicobacter pylori in Hispanics living in Puerto Rico: A population‐based study

Background

Helicobacter pylori is an important etiologic factor for peptic ulcers and gastric cancer, one of the top ten leading causes of cancer death in Puerto Rico. However, the prevalence of H. pylori infections in this population was previously unknown. The aim of this study was to examine the seroprevalence of H. pylori and its associated risk factors in Puerto Rico.

Materials and Methods

A cross‐sectional study was designed using an existing population‐based biorepository. Seropositivity was determined using the Premier^? H. pylori immunoassay. Helicobacter pylori seroprevalence was estimated with 95% confidence using marginal standardization following logistic regression. To assess the risk factors associated with H. pylori seropositivity, a multivariable log‐binomial model was fitted to estimate the prevalence ratio (PR) and its 95% confidence interval (95% CI).

Results

A total of 528 population‐based serum samples were analyzed. The mean age of the study population was 41 ± 12 years, of whom 55.3% were females. The overall seroprevalence of H. pylori was 33.0% (95% CI = 28.3%‐38.1%). Increasing age and having <12 years of education were significantly (P < .05) associated with H. pylori seropositivity in the multivariable model; however, residing in counties with low population density reached marginal significance (P = .085).

Conclusions

We report that H. pylori infection is common among Hispanics living in Puerto Rico. The H. pylori seroprevalence observed in Puerto Rico is similar to the seroprevalence reported in the overall population of the United States. The association between H. pylori seroprevalence and the risk factors analyzed offers insight into the epidemiology of gastric cancer in Puerto Rico and warrants further investigation.

     

Nano‐based methods for novel coronavirus 2019 (2019‐nCoV) diagnosis: A review

Today, tremendous attention has been devoted to a new coronavirus, SARS‐CoV‐2 (2019‐nCoV), due to severe effects on the global public in all over the world. Rapid and accurate diagnosis of 2019‐nCoV are important for early treatment and cutting off epidemic transmission. In this regard, laboratory detection protocols, such as polymerase chain reaction (PCR) and computed tomography (CT) examination, have been utilized broadly for 2019‐nCoV detection. Recently, nano‐based methods for 2019‐nCoV diagnoses are rapidly expanding and declaring comparable results with PCR and CT. In this review, recent advances in nano‐based techniques have been highlighted and compared briefly with PCR and CT as well‐known methods for 2019‐nCoV detection.     

Improved Detection of Citrus psorosis virus and Coat Protein‐Derived Transgenes in Citrus Plants: Comparison Between RT‐qPCR and TAS‐ELISA

Citrus is one of the most economically important fruit crops in the world. Citrus psorosis is a serious disease affecting mainly oranges and mandarins in Argentina and Uruguay. The causal agent is Citrus psorosis virus (CPsV), an ophiovirus with a tripartite ssRNA genome of negative polarity. The coat protein (CP), the most abundant viral protein in infected plants, has been used to detect CPsV by TAS‐ELISA, but only biological indexing, requiring 1 year, is the current and validated technique for diagnosis of citrus psorosis. In this study, a SYBR Green RT‐qPCR protocol was developed, with primers designed to the most conserved region of the cp gene. We tested their specificity and sensitivity in comparison with TAS‐ELISA. This RT‐qPCR was applied successfully to field samples from Argentina, to a variety of isolates from different countries maintained in the greenhouse, to young seedlings and old trees from a psorosis natural transmission plot, and to transgenic citrus expressing the cp gene of CPsV or a fragment thereof. This method allowed accurate quantification of viral titer and cp gene expression in transgenic plants, which could not be detected previously. The sensitivity and reliability of quantitative CPsV detection were improved with greater speed using commercial reagents, and the sensitivity was three orders of magnitude higher than that of TAS‐ELISA. All these data encourage its validation.     

Rapid detection of viable Legionella pneumophila in tap water by a qPCR and RT‐PCR‐based method

Aims

A molecular method for a rapid detection of viable Legionella pneumophila of all serogroups in tap water samples was developed as an alternative to the reference method (ISO). Legionellae are responsible for Legionnaires’ disease, a severe pneumonia in humans with high lethality.

Methods and Results

The developed method is based on a nutritional stimulation and detection of an increase in precursor 16S rRNA as an indicator for viability. For quantification, DNA was detected by qPCR. This method was compared to the ISO method using water samples obtained from public sports facilities in Switzerland. The sensitivity and specificity were 91 and 97%, respectively, when testing samples for compliance with a microbiological criterion of 1000 cell equivalents per l.

Conclusion

The new method is sensitive and specific for Leg. pneumophila and allows results to be obtained within 8 h upon arrival, compared to one week or more by the ISO method.

Significance and Impact of the Study

The method represents a useful tool for a rapid detection of viable Leg. pneumophila of all serogroups in water by molecular biology. It can be used as an alternative to the ISO method for official water analysis for legionellae and particularly when a short test time is required.