RNA isolation efficacy of commercial and modified conventional methods for Citrus tristeza virus and mRNA internal control amplification

Huma, Nesheman; Sahar, Shagufta; Iftikhar, Yasir; Azhar, Abid; Galani, Saddia

doi:10.2478/s11756-019-00405-w

Cited by 4 publications

(4 citation statements)

References 30 publications

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“…The results described in the literature corroborates with the one obtained here when the RNA from sweet potato tissues was extracted through the hot acid phenol and CTAB methods, since IbSDR1 showed relatively higher expression levels in tuberous roots and displayed a basal expression level in the other tissues (stem and leaves) (Figure 4). On the other hand, when RNA was isolated through the TRIzol method, the results showed that the RNA quality directly affected the reaction, inhibiting the PCR, similar to what has been reported in other studies, as observed by Huma et al (2020). In this study, where different RNA isolation methods were tested in citrus, low values for the A 260 /A 230 ratio directly affect the expression analysis, leading to the absence of amplification in the RT-PCR analysis.…”

Section: Rt-qpcrsupporting

confidence: 84%

“…Several studies have been performed in different plant species, such as citrus (Huma et al, 2020), brassica (Siles et al, 2020), cassava (Behnam et al, 2019), Phaseolus vulgaris (Acosta-Maspons et al, 2019), Elaeis guineensis (Ong et al, 2019), and coffee (Paula et al, 2012;Huded et al, 2018) aiming to establish the best method for obtaining high-quality RNA. Plant tissues may have compounds, such as polysaccharides and polyphenols, that can negatively interfere in isolating high-quality nucleic acids (Badai et al, 2020), turning this process many times unique for each plant tissue.…”

Section: Introductionmentioning

confidence: 99%

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Research Article Evaluation of extraction methods for obtaining high-quality RNA from sweet potato

Gonçalves¹,

Daúde²,

Souza³

et al. 2021

Genet. Mol. Res.

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Section: Rt-qpcrsupporting

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Research Article Evaluation of extraction methods for obtaining high-quality RNA from sweet potato

Gonçalves¹,

Daúde²,

Souza³

et al. 2021

Genet. Mol. Res.

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“…More specifically, the adoption of PCRbased assays in citrus pathogen detection plays a critical role in disease management programs monitoring the sanitary status of germplasm and propagative materials, nursery stock, and commercial plantings of citrus and other agriculturally significant crops (Bostock et al, 2014;Gergerich et al, 2015;Osman et al, 2015;Albrecht et al, 2020;Fuchs et al, 2021). While the protocols for the development and validation of PCR assays are focused primarily on primer and probe sequence design, conditions, composition and efficiency of the reaction as well as the specificity, sensitivity, transferability and robustness of the PCR assay (Bustin et al, 2009;Broeders et al, 2014;Tan et al, 2019), the importance of the quality of nucleic acids isolated from a specific type of plant tissue that is used in the PCR assays cannot be overlooked (Fleige and Pfaffl, 2006;Becker et al, 2010;Taylor et al, 2010;Die and Romań, 2012;Huma et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

A comparative analysis of RNA isolation methods optimized for high-throughput detection of viral pathogens in California’s regulatory and disease management program for citrus propagative materials

Dang

Bodaghi²,

Osman³

et al. 2022

Front. Agron.

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Citrus germplasm programs can benefit from high-throughput polymerase chain reaction (PCR)-based methods for the detection of graft-transmissible pathogens in propagative materials. These methods increase diagnostic capacity, and thus contribute to the prevention of disease spread from nurseries to citrus orchards. High quality nucleic acids, as determined by purity, concentration, and integrity, are a prerequisite for reliable PCR detection of citrus pathogens. Citrus tissues contain high levels of polyphenols and polysaccharides, which can affect nucleic acid quality and inhibit PCR reactions. Various commercially available RNA isolation methods are used for citrus and include: phenol-chloroform (TRIzol®, Thermo Fisher Scientific); silica columns (RNeasy® Plant Mini Kit, Qiagen); and magnetic beads-based methods (MagMAX™-96 Viral RNA Isolation Kit, Thermo Fisher Scientific). To determine the quality of RNA and its impact on the detection of graft-transmissible citrus pathogens in reverse transcription (RT) PCR-based assays, we compared these three RNA isolation methods. We assessed RNA purity, concentration, and integrity from citrus inoculated with different viruses and viroids. All three RNA isolation methods produced high quality RNA, and its use in different RT-PCR assays resulted in the detection of all targeted citrus viruses and viroids with no false positive or negative results. TRIzol® yielded RNA with the highest concentration and integrity values but some samples required serial dilutions to remove PCR inhibitors and detect the targeted pathogens. The RNeasy® kit produced the second highest concentration and purity of RNA, and similar integrity to TRIzol®. MagMAX™ isolation also provided high quality RNA but most importantly produced RNA with consistent results clustered around a median value for concentration, purity, and integrity. Subsequently, MagMAX™-96 was combined with the semi-automated MagMAX™ Express-96 Deep Well Magnetic Particle Processor, for high-throughput sample processing. MagMAX™-96 enabled the diagnostic laboratory of the Citrus Clonal Protection Program-National Clean Plant Network at the University of California, Riverside to process over 16,500 samples from citrus budwood source trees between 2010 and 2019. This high-throughput approach dramatically reduced the incidence of viroids in citrus nurseries and was key to the successful implementation of the mandatory Citrus Nursery Stock Pest Cleanliness Program in California.

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“…RNA was isolated from cell by using TRIzol method. 11 The PCR tested samples were seen on gel doc system to see the bands of DNA. 12…”

Section: Methodsmentioning

confidence: 99%

Frequency of Thalassemia and Its Association with Hepatitis C Infection in Local Population of Pakistan

Khan¹,

Reyaz²,

Baloch³

et al. 2020

BIOMEDICA

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Background and Objective: Thalassemia major or βeta-thalassemia is a hereditary blood disorder, necessitating continuous blood transfusions. Frequent blood transfusions often result in the development of hepatitis, hepatocellular carcinoma, acquired immunodeficiency syndrome (AIDS) and many other infectious diseases. Incidence of infection of viral hepatitis in thalassemia patients is majorly associated with infections in the donor population. The objective of this study was to ascertain the frequency of HCV infection in patients with thalassemia in Pakistan.Methods: This study was conducted from January 2015 to September 2018 in various sections of Punjab and Sindh, Pakistan. A total number of 439 patients were enrolled in this study, samples of blood from the patients were collected and their Anti-HCV antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA); HCV-RNA genome presentation was assessed by HCV-nested-real time-polymerase chain reaction (RT-PCR) for the confirmation.Results: It was found that major population of the thalassemia patients were male (63%). While the female thalassemia population was almost twice in Sindh province as compared to Punjab. In present study, n = 187 were detected positive among those n = 168 were confirmed by PCR. Only 47% thalassemia patients were found vaccinated against hepatitis B whereas all HCV positive patients were not vaccinated, and (84.96%) thalassemia patients were from the family of first cousin marriage. The patients which are not vaccinated also had significant chance to acquire the viral infection (P < 0.001). Moreover there could be some gender and environmental or geographical factors associated with thalassemia that are yet to be explored in detail. Conclusion:Thalassemia patients carry major risk for acquiring transfusion associated infection.

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RNA isolation efficacy of commercial and modified conventional methods for Citrus tristeza virus and mRNA internal control amplification

Cited by 4 publications

References 30 publications

Research Article Evaluation of extraction methods for obtaining high-quality RNA from sweet potato

Research Article Evaluation of extraction methods for obtaining high-quality RNA from sweet potato

A comparative analysis of RNA isolation methods optimized for high-throughput detection of viral pathogens in California’s regulatory and disease management program for citrus propagative materials

Frequency of Thalassemia and Its Association with Hepatitis C Infection in Local Population of Pakistan

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