A new real-time PCR assay for detecting fungi in genus Ceratocystis

Plant Disease ◽  
2021 ◽  
Author(s):  
Karthikeyan Dharmaraj ◽  
Alice Merrall ◽  
Julie A. Pattemore ◽  
Joanne Mackie ◽  
Brett J.R Alexander ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Pathogens ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Stem Tissue ◽  
Natural Ecosystems ◽  
Pcr Assay ◽  
Practical Applications ◽  
Wide Range

The genus Ceratocystis contains several significant plant pathogens, causing wilt and canker disease on a wide range of plants species. Currently, there are over 40 known species of Ceratocystis, some of which are becoming increasingly important in agricultural or natural ecosystems. The diagnostics for most Ceratocystis species currently relies on time consuming and labour-intensive culturing approaches. To provide more time efficient and sensitive molecular diagnostic tools for Ceratocystis, a generic Taq-Man real-time PCR assay was developed using the ITS gene. This novel two-probe Taq-man assay amplified DNA from all tested Ceratocystis species. Some non-specific amplification of a few species from closely related genera was observed under certain conditions; however, these false positive detections could be ruled out using the additional PCR primers developed for further sequence based identification of the detected species. The assay was highly sensitive as it detected 0.2 pg/µl of Ceratocystis DNA in water as well as in host DNA matrix. Further validation with artificially inoculated fig stem tissue demonstrated that the assay was also able to effectively detect the pathogen in infected asymptomatic stem tissue. This newly developed real-time PCR assay has practical applications in biosecurity, conservation, and agriculture, enabling to detect Ceratocystis species directly from plant material, to facilitate more sensitive screening of imported plant germplasm, and allow rapid tracking of pathogens in case of disease outbreaks.

scholarly journals Use of Multiplex Real-Time PCR To Diagnose Scrub Typhus

2017 ◽  
Vol 55 (5) ◽  
pp. 1377-1387 ◽  
Author(s):  
Wiwit Tantibhedhyangkul ◽  
Ekkarat Wongsawat ◽  
Saowaluk Silpasakorn ◽  
Duangdao Waywa ◽  
Nuttawut Saenyasiri ◽  
...  
Keyword(s):  
Early Diagnosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Scrub Typhus ◽  
Febrile Illness ◽  
Asia Pacific ◽  
Diagnostic Tools ◽  
Pcr Assay ◽  
Content Type ◽  
Serologic Tests

ABSTRACTScrub typhus, caused byOrientia tsutsugamushi, is a common cause of acute undifferentiated febrile illness in the Asia-Pacific region. However, its nonspecific clinical manifestation often prevents early diagnosis. We propose the use of PCR and serologic tests as diagnostic tools. Here, we developed a multiplex real-time PCR assay using hydrolysis (TaqMan) probes targetingO. tsutsugamushi47-kDa,groEL, and human interferon beta (IFN-β gene) genes to improve early diagnosis of scrub typhus. The amplification efficiency was higher than 94%, and the lower detection limit was 10 copies per reaction. We used a human gene as an internal DNA quality and quantity control. To determine the sensitivity of this PCR assay, we selected patients with confirmed scrub typhus who exhibited a clear 4-fold increase in the level of IgG and/or IgM. The PCR assay result was positive in 45 of 52 patients, indicating a sensitivity of 86.5% (95% confidence interval [CI]: 74.2 to 94.4). The PCR assessment was negative for all 136 non-scrub typhus patients, indicating a specificity of 100% (95% CI: 97.3 to 100). In addition, this test helped diagnose patients with inconclusive immunofluorescence assay (IFA) results and using single blood samples. In conclusion, the real-time PCR assay proposed here is sensitive and specific in diagnosing scrub typhus. Combining PCR and serologic tests will improve the diagnosis of scrub typhus among patients presenting with acute febrile illness.

Improving the specific diagnosis of trematode, cestode and nematode infections by a multiplex single-tube real-time PCR assay

2019 ◽  
Vol 72 (7) ◽  
pp. 487-492 ◽  
Author(s):  
Samson S Y Wong ◽  
Rosana W S Poon ◽  
Kelvin K W To ◽  
Jasper F W Chan ◽  
Gang Lu ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Molecular Diagnostic ◽  
Rrna Gene ◽  
28S Rrna ◽  
Pcr Assay ◽  
Single Tube ◽  
Histological Sections ◽  
Pcr Products ◽  
Stool Samples

AimsHelminth infections are becoming uncommon in high-income countries and laboratory staff may lose expertise in their morphological identification, especially in histological sections where speciation of helminths is challenging. Commercially available molecular diagnostic panels for faecal specimens only offer tests for protozoa but not helminths. We aim to improve the identification accuracy of helminths using a multiplex PCR assay.MethodsWe designed three pairs of PCR primers and probes targeting multicopy genes for a multiplex single-tube real-time PCR assay which covers 16 trematode (28S rRNA gene), 24 cestode (cox1 gene) and 33 nematode (cox1 gene) species. Helminths (n=27) from faecal samples (n=10), fresh parasites (n=11), formalin-fixed specimens (n=4), cerebrospinal fluid (n=1) and bile (n=1) were examined morphologically and tested by PCR. Fifty stool samples negative for parasites by microscopy were also tested.ResultsThe PCR assay correctly identified the genera of all tested helminths. Agarose gel electrophoresis and sequencing of the purified PCR amplicons confirmed that the PCR products were of correct sizes with 100% correlation with the respective species. Sequencing of the cox1 gene failed to identify Capillaria spp. in one sample owing to the lack of corresponding sequences in GenBank. PCR and sequencing of the nematode 18S rRNA gene using consensus primers showed 100% homology with Capillaria spp. sequence. No positive PCR products were found in the negative stool samples.ConclusionsThe highly specific test correctly identified all helminths in our cohort. It is a useful adjunct to helminth identification in difficult situations such as histological sections.

Evaluation of Three Real-Time PCR Methods for Detection of Salmonella from Cloves

2017 ◽  
Vol 80 (6) ◽  
pp. 982-989 ◽  
Author(s):  
Aparna Tatavarthy ◽  
Laila Ali ◽  
Vikas Gill ◽  
Lijun Hu ◽  
Xiaohong Deng ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Extraction Methods ◽  
Pcr Assay ◽  
Wide Range ◽  
The Mean ◽  
Salmonella Serotypes ◽  
Pathogen Dna ◽  
Dna Extraction Methods

ABSTRACTThe purpose of the study was to evaluate three real-time PCR platforms for rapid detection of Salmonella from cloves and to compare three different DNA extraction methods. Six trials were conducted with two clove cultivars, Ceylon and Madagascar, and three Salmonella serotypes, Montevideo, Typhimurium, and Weltevreden. Each trial consisted of 20 test portions. The preenrichment cultures were used to perform PCR for comparison of the effectiveness of U.S. Food and Drug Administration, Pacific Regional Laboratory Southwest (FDA-PRLSW), Applied Biosystems Inc. (ABI) MicroSEQ, and GeneDisc platforms for detection of Salmonella. Three DNA extraction methods were used: standard extraction method for each PCR platform, boil preparation, and LyseNow food pathogen DNA extraction cards. The results from real-time PCR correlated well with FDA Bacteriological Analytical Manual culture assay results, with a wide range of cycle threshold (CT) values among the three PCR platforms for intended positive samples. The mean CT values for MicroSEQ (16.36 ± 2.78) were significantly lower than for PRLSW (20.37 ± 3.45) and GeneDisc (23.88 ± 2.90) (P < 0.0001). Pairwise comparisons between PCR platforms using different DNA extraction methods indicate that the CT values are inversely proportional to the relative DNA quantity (RDQ) yields by different platform-extraction combinations. The pairing of MicroSEQ and boil preparation generated the highest RDQ of 120 and the lowest average CT value of 14.48, whereas the pairing of GeneDisc and LyseNow generated the lowest RDQ of 0.18 and the highest average CT of 25.97. Boil preparation yielded higher RDQ than the other extraction methods for all three PCR platforms. Although the MicroSEQ platform generated the lowest CT values, its sensitivity was compromised by narrow separations between the positive and negative samples. The PRLSW platform generated the best segregation between positive and negative groups and is less likely to produce false results. In conclusion, FDA-PRLSW was the most efficient PCR assay for Salmonella detection, and boil preparation was the best method for DNA extraction.

scholarly journals New Subgenotyping and Consensus Real-Time Reverse Transcription-PCR Assays for Hepatitis A Outbreak Surveillance

2019 ◽  
Vol 57 (9) ◽  
Author(s):  
William S. Probert ◽  
Jill K. Hacker
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Outbreak Detection ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Laboratory Surveillance

ABSTRACTLaboratory surveillance plays an important role in the detection and control of hepatitis A outbreaks and requires the application of rapid and accurate molecular diagnostic tools for hepatitis A virus (HAV) RNA detection, subgenotype identification, and sequence-based genotyping. We describe the development and validation of a triplex real-time, reverse transcription-PCR (triplex rRT-PCR) assay for the identification and discrimination of HAV subgenotypes IA, IB, and IIIA and a singleplex rRT-PCR assay designed to detect all HAV genotypes infecting humans. Overall, the accuracy, sensitivity, and specificity of the new assays were >97% for serum and plasma specimens collected during unrelated outbreaks of HAV in California and Michigan compared to a nested RT-PCR genotyping assay and the ISO 15216-1 rRT-PCR method for HAV detection. The new assays will permit the rapid detection of HAV RNA and discrimination among subgenotypes IA, IB, and IIIA in serum and plasma specimens, which will strengthen public health surveillance efforts for HAV outbreak detection and response.

Development of a Quantitative Real-Time PCR Method To Enumerate Total Bacterial Counts in Ready-to-Eat Fruits and Vegetables

2006 ◽  
Vol 69 (10) ◽  
pp. 2504-2508 ◽  
Author(s):  
HAJIME TAKAHASHI ◽  
HIROTAKA KONUMA ◽  
YUKIKO HARA-KUDO
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fruits And Vegetables ◽  
Bacterial Species ◽  
Culture Method ◽  
Plate Count ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Cycle Number ◽  
Wide Range

A newly developed real-time PCR assay rapidly quantifies the total bacterial numbers in contaminated ready-to-eat vegetables and fruits compared with the standard plate count method. Primers targeting the rpoB gene, which encodes for the β subunit of the bacterial RNA polymerase and which is common to most bacterial species, was used instead of the 16S rRNA gene, which has multiple copies and varies among bacterial species. A primer pair specific for rpoB was confirmed to amplify rpoB in a wide range of bacterial species after we assessed 49 strains isolated from five kinds of fruits and vegetables. We purchased fruits and vegetables from retail shops and enumerated the bacteria associated with them by use of real-time PCR and compared this to the number found by the culture method. We found a high correlation between the threshold PCR cycle number when compared with the plate count culture number. The real-time PCR assay developed in this study can enumerate the dominant bacterial species in ready-to-eat fruits and vegetables.

scholarly journals Evaluation of digital real-time PCR assay as a molecular diagnostic tool for single-cell analysis

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Chia-Hao Chang ◽  
Daxen Mau-Hsu ◽  
Ke-Cheng Chen ◽  
Cheng-Wey Wei ◽  
Chiung-Ying Chiu ◽  
...  
Keyword(s):  
Single Cell ◽  
Real Time ◽  
Diagnostic Tool ◽  
Real Time Pcr ◽  
Single Cell Analysis ◽  
Molecular Diagnostic ◽  
Cell Analysis ◽  
Pcr Assay

scholarly journals Rapid Detection and Differentiating of the Predominant Salmonella Serovars in Chicken Farm by TaqMan Multiplex Real-Time PCR Assay

2021 ◽  
Vol 11 ◽  
Author(s):  
Suhua Xin ◽  
Hong Zhu ◽  
Chenglin Tao ◽  
Beibei Zhang ◽  
Lan Yao ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Epidemiologic Study ◽  
Clinical Diagnostics ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Chicken Farm

Salmonella has been known as an important zoonotic pathogen that can cause a variety of diseases in both animals and humans. Poultry are the main reservoir for the Salmonella serovars Salmonella Pullorum (S. Pullorum), Salmonella Gallinarum (S. Gallinarum), Salmonella Enteritidis (S. Enteritidis), and Salmonella Typhimurium (S. Typhimurium). The conventional serotyping methods for differentiating Salmonella serovars are complicated, time-consuming, laborious, and expensive; therefore, rapid and accurate molecular diagnostic methods are needed for effective detection and prevention of contamination. This study developed and evaluated a TaqMan multiplex real-time PCR assay for simultaneous detection and differentiation of the S. Pullorum, S. Gallinarum, S. Enteritidis, and S. Typhimurium. In results, the optimized multiplex real-time PCR assay was highly specific and reliable for all four target genes. The analytical sensitivity corresponded to three colony-forming units (CFUs) for these four Salmonella serovars, respectively. The detection limit for the multiplex real-time PCR assay in artificially contaminated samples was 500 CFU/g without enrichment, while 10 CFU/g after pre-enrichment. Moreover, the multiplex real-time PCR was applied to the poultry clinical samples, which achieved comparable results to the traditional bacteriological examination. Taken together, these results indicated that the optimized TaqMan multiplex real-time PCR assay will be a promising tool for clinical diagnostics and epidemiologic study of Salmonella in chicken farm and poultry products.

scholarly journals TaqMan quantitative real-time PCR for detecting Avipoxvirus DNA in various sample types from hummingbirds

2020 ◽  
Author(s):  
Hanna E. Baek ◽  
Ravinder N. Sehgal ◽  
Ruta R. Bandivadekar ◽  
Pranav Pandit ◽  
Michelle Mah ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Disease Ecology ◽  
Core Protein ◽  
Bird Species ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Wide Range ◽  
Avian Pox ◽  
The Impact

AbstractBackgroundAvian pox is a viral disease documented in a wide range of bird species. Disease related detrimental effects can cause dyspnea and dysphagia, therefore birds with high metabolic requirements, such as hummingbirds, are especially vulnerable. Hummingbirds have a strong presence in California, especially in urban environments; however, little is understood regarding the impact of pox virus on hummingbird populations. Diagnosing pox infections relies on obtaining a tissue biopsy that poses significant bird risks and field challenges. Understanding the ecology of hummingbird pox viral infections could be advanced by a minimally invasive ante-mortem diagnostic method. This study’s goal was to address this gap in understanding if pox infections can be diagnosed using integumentary system samples besides tissue biopsies. To meet this goal, we tested multiple integumentary sample types and tested them using a quantitative real-time PCR assay. A secondary study goal was to determine which sample types (ranging from minimally to highly invasive sampling) were optimal for identifying infected birds.Methodology/Principal FindingsLesion tissue, pectoral muscle, feathers, toenail, blood, and swabs (both lesion tissue and non-lesion tissues) were taken from live birds and carcasses of two species of hummingbirds found in California. To maximize successful diagnosis, especially for samples with low viral load, a real-time quantitative PCR assay was developed for detecting the hummingbird-specific Avipoxvirus 4b core protein gene. Avipoxvirus DNA was successfully amplified from all sample types across 27 individuals. Our results were then compared to those of conventional PCR. Comparisons were also made between sample types utilizing lesion tissue samples as the gold standard.Conclusions/SignificanceHummingbird avian pox can be diagnosed without relying on tissue biopsies. Feather samples can be used for diagnosing infected birds and reduces sampling risk. A real-time PCR assay detected viral DNA in various integumentary system sample types and could be used for studying hummingbird disease ecology in the future.

QUANTITATION OF HBV DNA BY REALTIME PCR FOR HBV DETECTION AND FOLLOW-UP VIRAL LOAD IN PATIENTS WITH CHRONICHBV HEPATITIS USING ANTIVIRAL DRUGS

2011 ◽  
pp. 64-70
Author(s):  
Van An Le ◽  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Antiviral Drugs ◽  
Hbv Dna ◽  
Therapeutic Effects ◽  
Pcr Assay ◽  
Serum Samples ◽  
Wide Range ◽  
Chronic Hbv

Background: Real-time PCR assay has been routinely used in many laboratories for HBV determination and follow –up of the HBV DNA levels in serum of chronic HBV patients during antiviral therapy. We used a commercialized real-time PCR procedure based on TaqMan chemistry to quantify HBV DNA levels from patients with chronic HBV hepatitis for HBV detection and monitoring HBV DNA during antiviral drug using. Materials and method: This study was carried out in 56 patients with chronic HBV hepatitis using antiviral drugs. A commercialized real-time PCR assay based on TaqMan chemistry was used to quantify HBV DNA concentration in double serum samples from each patient, the first sample was collected at the first quantificative testing and the second sample was collected for a follow-up in 6 or 9 months of interval. The assay has a dymnamic range from 3 x 102 copies/ml at minimun level to 1010copies /ml. Sample testing was always run with triple dilutions of standard, the HBV DNA quantitations were analysed by Stratagene software and calculated in number of copies per ml of serum sample. Results: The HBV DNA levels in all the first serum samples had a wide range from 3x102 to 1010 copies/ml, of these first samples there were 30% (17/56) with the highest levels from 108 to 1010copies/ml and there was no sample negative for HBV DNA. With the second serum samples, there were 23,2% (13/56) underdetectable for HBV DNA and the sample percentage with the highest HBV DNA levels was only 5,3% (3/56). The HBV DNA levels at the second serum samples were lower in 44 patients (78%) and were higher in 12 patients (21,4%) in comparision with that of the first samples. The average amounts of HBV DNA decrease in patients using antiviral drugs were 2,3 x 108 copies/ml with adefovir and 4,2 x108 copies/ml with lamivudine, and the average numbers of HBV DNA increase were 1,4 x 108 copies/ml with lamivudine and 7,5 x 107 copies/ml with entecavir. Conclusions: Real-time PCR assay was found to be very useful in quantification of HBV DNA level in chronic HBV patients and also for monitoring the therapeutic effects of antiviral drugs.

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