Real-time PCR as a promising tool to monitor growth of Venturia spp. in scab-susceptible and -resistant apple leaves

The methanogenic community structures of four different anaerobic processes were characterized using a quantitative real-time PCR with group-specific primer and probe sets targeting the 16S rRNA gene (rDNA). The group specific primer and probe sets were developed and used to detect the orders Methanosarcinales, and the families Methanosarcinaceae and Methanosaetaceae. Two separate sets targeting the domains Archaea and Bacteria were also used. Each microbial population in different anaerobic processes was determined and the relative abundance in the system was compared with each other. Dominant methanogenic populations and the community structures in the processes were varied by hydraulic retention time and acetate concentration. This indicates that the real-time PCR method with the primer and probe sets is a promising tool to analyze community structures in anaerobic processes.

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Validation of an in-house real-time PCR fecal assay and comparison with two commercial assays for the antemortem detection of Mycobacterium avium subsp. paratuberculosis infection in culled sheep

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638718810744 ◽

2018 ◽

Vol 31 (1) ◽

pp. 58-68 ◽

Cited By ~ 1

Author(s):

Julie Arsenault ◽

Jagdip Singh Sohal ◽

Anne Leboeuf ◽

Pierre Hélie ◽

Gilles Fecteau ◽

...

Keyword(s):

Real Time ◽

Mycobacterium Avium ◽

Real Time Pcr ◽

Positive Culture ◽

Cost Effective ◽

Mycobacterium Avium Subsp ◽

Fecal Culture ◽

Convenience Sample ◽

Promising Tool ◽

Mycobacterium Avium Subsp Paratuberculosis

Paratuberculosis is a chronic infectious enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). In sheep, the antemortem detection of the infection is challenging given the slow progression of the disease and the lack of sensitive, specific, and cost-effective validated tests. We adapted an in-house real-time PCR (rtPCR) assay targeting the multi-copy IS 900 element of MAP. The sensitivity and specificity of this essay for the detection of MAP infection were estimated in a convenience sample of culled ewes from 7 infected flocks and compared to a commercial fecal rtPCR, a commercial ELISA, and fecal culture. An infected ewe was defined as a ewe with a positive culture of the ileum and/or mesenteric lymph node. A non-infected ewe was defined as a ewe negative in intestinal tissue culture, negative in fecal culture, and with no lesions consistent with paratuberculosis. The in-house rtPCR had a sensitivity estimate of 84% (95% confidence interval [CI]: 59%, 97%) among the 44 infected ewes, which was significantly higher ( p ⩽ 0.05) than the sensitivity of a commercial fecal rtPCR (52%, 95% CI: 27%, 76%; or 63%, 95% CI: 35%, 87% depending on the cutoff used), an ELISA (14%, 95% CI:2.0%, 41%), and fecal culture (21%, 95% CI: 2.7%, 59%). No statistical difference in assay specificities was observed for the 30 non-infected ewes. The in-house rtPCR is a promising tool that could be used advantageously for the antemortem detection of MAP infection in sheep.

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Molecular mechanism behind the involvement of apple flavonoid glycosyltransferase gene MdGT1 in the color of apple leaves

10.1101/2021.02.01.429094 ◽

2021 ◽

Author(s):

Pan Li ◽

Hongjuan Ge ◽

Lei Zhang ◽

Jing Shu ◽

Zhuojing Sun ◽

...

Keyword(s):

Transcription Factor ◽

Real Time ◽

Mutant Strain ◽

Real Time Pcr ◽

Physiological Data ◽

Plant Tolerance ◽

Mobility Shift ◽

Glycosyltransferase Gene ◽

Apple Leaves

SummaryFlavonoids are a class of polyphenol compounds that are widespread in plants. They play an important role in plant growth and development. In this study, we found a mutant strain of M. baccata with yellow leaves (YL). Transcriptome sequencing revealed that it exhibited significant changes in the flavonoid metabolism pathway, which screening revealed was associated with a glycosyltransferase gene, MD09G1064900 (MdGT1). Analysis of its spatiotemporal expression showed that MdGT1 was mainly expressed in the stem and leaves, it means that MdGT1 may have a functional role in these parts. Real-time PCR and HPLC showed that MdGT1 was significantly upregulated by anthocyanin and exhibited strong anthocyaninase activity in vitro, respectively. An MdGT1 plant expression vector was constructed and overexpressed in apple fruit callus, resulting in a significant decrease of anthocyanin. Phenotypic observation also revealed that the MdGT1-overexpressing lines exhibited worse growth than the wild type after NaCl treatment, while they grew better upon the addition of exogenous anthocyanins. Moreover, real-time PCR and physiological data showed that MdGT1 is involved in salt stress and closely related to antioxidant pathways. Electrophoretic mobility shift assays (EMSA) and yeast one-hybrid experiments also proved that the transcription factor MdMYB88 is an upstream regulatory factor of MdGT1. The sequencing results revealed an amino acid insertion in an MdMYB88 HTH domain (between 77-131 amino acids) in the YL mutant strain. In conclusion, we identified a new apple glycosyltransferase gene, MdGT1, which may affect the color of apple leaves by glycosylating anthocyanins, and be regulated by the upstream transcription factor MdMYB88.Significance statementThe glycosyltransferases and their physiological significance in apple are largely unknown. Here we revealed that the MdMYB88-regulated apple glycosyltransferase gene MdGT1 plays a crucial role in the color of apple leaves and enhances plant tolerance to salt by antioxidant pathways via anthocyanin metabolism.

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Evaluation of Real-time PCR for Diagnosis of Post-Kala-azar Dermal Leishmaniasis in Endemic Foci of Bangladesh

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy234 ◽

2018 ◽

Vol 5 (10) ◽

Cited By ~ 4

Author(s):

Prakash Ghosh ◽

Md Golam Hasnain ◽

Faria Hossain ◽

Md Anik Ashfaq Khan ◽

Rajashree Chowdhury ◽

...

Keyword(s):

Real Time ◽

Clinical Examination ◽

Real Time Pcr ◽

Potential Method ◽

Diagnostic Methods ◽

Promising Tool ◽

Kala Azar ◽

Polymerase Chain ◽

Strip Test ◽

Treatment Prognosis

Abstract Background Post-kala-azar dermal leishmaniasis (PKDL) is a sequel to visceral leishmaniasis (VL), which is found in VL-endemic countries including Bangladesh. Because of these enigmatic cases, the success of the National Kala-azar Elimination Program is under threat. To date, diagnostic methods for PKDL cases in endemic regions have been limited to clinical examination and rK39 test or microscopy, and a suitable and accurate alternative method is needed. In this study, we investigated the application of real-time polymerase chain reaction (PCR) as a potential method for diagnosis of PKDL in comparison with microscopy. Methods Ninety-one suspected macular PKDL cases from Mymensingh district, Bangladesh, were enrolled in the study after diagnosis by clinical examination and an rK39 strip test. All of them responded after completion of the treatment with miltefosine. During enrollment, a skin biopsy was done for each patient, and both microscopy and real-time PCR were performed for detection and quantification of Leishmania donovan body (LDB) and LD DNA, respectively. Results Real-time PCR detected 83 cases among all suspected PKDL patients, with an encouraging sensitivity of 91.2% (83.4%–96.1%), whereas microscopy showed 50.6% (39.9%–61.2%) sensitivity. Among all suspected PKDL cases, 42 cases were positive in both microscopy and qPCR, whereas 41 cases were detected as positive through qPCR only. Conclusions This study provides evidence that real-time PCR is a promising tool for diagnosis of PKDL in endemic regions. In addition to diagnosis, the quantitative ability of this method could be further exploited for after-treatment prognosis and cure assessment of PKDL cases.

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Quantitative real-time PCR as a promising tool for the detection and quantification of leaf-associated fungal species – A proof-of-concept using Alatospora pulchella

PLoS ONE ◽

10.1371/journal.pone.0174634 ◽

2017 ◽

Vol 12 (4) ◽

pp. e0174634 ◽

Cited By ~ 6

Author(s):

Alexander Feckler ◽

Anne Schrimpf ◽

Mirco Bundschuh ◽

Felix Bärlocher ◽

Patrick Baudy ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Fungal Species ◽

Proof Of Concept ◽

Quantitative Real Time Pcr ◽

Promising Tool ◽

Detection And Quantification

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Detection of SARS-CoV-2 in the sewerage system in Tunisia: a promising tool to confront COVID-19 pandemic

Future Virology ◽

10.2217/fvl-2021-0050 ◽

2021 ◽

Author(s):

Habib Jmii ◽

Hakima Gharbi-Khelifi ◽

Raouia Assaoudi ◽

Mahjoub Aouni

Keyword(s):

Quantitative Analysis ◽

Real Time ◽

Real Time Pcr ◽

Viral Rna ◽

Sewerage System ◽

Promising Tool ◽

Elution Method ◽

Second Period

Aim: The current study undertaken in Tunisia examines the use of wastewaters to monitor SARS-CoV-2 circulation. Materials & methods: Viral genetic materials collected in wastewaters during two different periods (September–October 2020 and February–April 2021) were concentrated using the adsorption-elution method. SARS-CoV-2 genes were researched by real-time PCR. Results: During the first period of the study, viral RNA was detected in 61.11% of the analyzed samples collected from Monastir city with a rate of 88.88% for raw wastewaters and 33.33% for treated wastewaters. Then, during the second period of the study, the quantitative analysis of wastewaters collected from seven governorates showed the presence of viral RNA among around 25% of them with variable RNA loads. The increased amounts of viral RNA detected in wastewaters were accompanied by an increase in the number of COVID-19 patients in Tunisia. Conclusion: Our results emphasize the importance of sewage survey in SARS-CoV-2 tracking.

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Detection and Quantification of Fusarium spp. (F. oxysporum, F. verticillioides, F. graminearum) and Magnaporthiopsis maydis in Maize Using Real-Time PCR Targeting the ITS Region

Agronomy ◽

10.3390/agronomy9020045 ◽

2019 ◽

Vol 9 (2) ◽

pp. 45 ◽

Cited By ~ 4

Author(s):

Maria Campos ◽

Mariana Patanita ◽

Catarina Campos ◽

Patrick Materatski ◽

Carla Varanda ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Fungal Pathogens ◽

High Sensitivity ◽

High Specificity ◽

Its Region ◽

Ear Rot ◽

Fusarium Spp ◽

Promising Tool ◽

Pcr Targeting

Fusarium spp. and Magnaporthiopsis maydis are soil-inhabiting fungi and respectively the causal agents of fusarium ear rot and late wilt, two important diseases that can affect maize, one of the most important cereal crops worldwide. Here, we present two sensitive real-time PCR TaqMan MGB (Minor Groove Binder) assays that detect and discriminate several Fusarium spp. (F. oxysporum, F. verticillioides, and F. graminearum) from M. maydis. The method is based on selective real-time qPCR amplification of the internal transcribed spacer (ITS) region and allows the quantification of the fungi. The applicability of this newly developed TaqMan methodology was demonstrated in a field experiment through the screening of potentially infected maize roots, revealing a high specificity and proving to be a suitable tool to ascertain Fusarium spp. and M. maydis infection in maize. Its high sensitivity makes it very efficient for the early diagnosis of the diseases and also for certification purposes. Thus, qPCR through the use of TaqMan probes is here proposed as a promising tool for specific identification and quantification of these soil-borne fungal pathogens known to cause disease on a large number of crops.

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