Application of Conventional PCR and Real-Time PCR Diagnostic Methods for Detection of the PineWood Nematode, Bursaphelenchus xylophilus, in Wood Samples from Lodgepole Pine

Abstract INTRODUCTION: Infection with Mycoplasma pneumoniae (M. pneumonia) occurs worldwide which accounts for 15%–20% of cases of community-acquired pneumonia and indistinguishable clinically from other infectious causes of pneumonia. AIM: The aim of this study was to evaluate the real-time polymerase chain reaction (PCR) and to correlate it with other diagnostic methods such as culture, serology (ELISA), and conventional PCR along with the clinical signs and symptoms produced by M. pneumonia. MATERIALS AND METHODS: A total of 130 patients of all age groups presenting with clinical features of lower respiratory tract infections were enrolled over a period of 1 year and 2 months in a tertiary care hospital in Delhi. M. pneumoniae in throat swab samples was detected by real-time PCR, compared with culture, serology, conventional PCR, and clinical signs and symptoms. Univariate analyses were conducted to determine the association of M. pneumoniae infection among different categories of patients. RESULTS: Out of a total of 130 patients, 18 patients (14%) were positive for M. pneumoniae by any test; culture was positive in nine patients (50%), serology (IgM) in eight patients (44.4%), PCR in five patients (27.7%), and real-time PCR was positive in six patients (33.3%). Clinical signs and symptoms were higher in incidence in M. pneumoniae-positive patients. Age-matched healthy controls (30) were included in the study, and all were negative for any diagnostic test performed (P = 0.026). CONCLUSION: It was concluded that combination of M. pneumoniae-specific testing modalities is required for the diagnosis of this etiological agent rather than a single diagnostic method.

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Application of a real-time PCR method for the detection of pine wood nematode, Bursaphelenchus xylophilus, in wood samples from lodgepole pine

Nematology ◽

10.1163/156854107781352098 ◽

2007 ◽

Vol 9 (3) ◽

pp. 351-362 ◽

Cited By ~ 41

Author(s):

Margaret Green ◽

Michael Rott ◽

Isabel Leal ◽

Leland Humble ◽

Eric Allen

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pinus Contorta ◽

Lodgepole Pine ◽

Nematode Species ◽

Bursaphelenchus Xylophilus ◽

Pine Wood Nematode ◽

Pine Wood ◽

Pure Cultures ◽

Pcr Method

AbstractA real-time PCR polymerase chain reaction (real-time PCR) method was developed to detect and differentiate Bursaphelenchus xylophilus (pine wood nematode, PWN) from other wood-inhabiting nematode species. A primer set and a specific Taqman® fluorescent probe were designed to amplify target B. xylophilus heat shock protein 70 sequences. After optimization, this real-time PCR assay was shown to be highly specific and sensitive, detecting at least 0.005 ng of B. xylophilus genomic DNA, as well as DNA extracted from single nematodes. The practical application of this real-time PCR diagnostic method for the detection of B. xylophilus from actual wood samples of lodgepole pine (Pinus contorta, Dougl. var. latifolia) trees containing a heterogeneous population of nematodes, rather than pure cultures or individual nematodes, is demonstrated. This method works well in the presence of potential inhibitors associated with wood after Baermann extraction and thus eliminates the need to produce pure nematode samples through further culturing and/or isolation of nematodes with a high-power microscope.

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DEVELOPMENT OF MULTIPLEX REAL-TIME PCR ASSAY FOR SIX PATHOGENS: RAPID TOOL FOR DIAGNOSIS OF BACTERIAL MENINGITIS

Journal of Medicine and Pharmacy ◽

10.34071/jmp.2019.3.8 ◽

2019 ◽

pp. 60-66

Author(s):

Viet Quynh Tram Ngo ◽

Thi Ti Na Nguyen ◽

Hoang Bach Nguyen ◽

Thi Tuyet Ngoc Tran ◽

Thi Nam Lien Nguyen ◽

...

Keyword(s):

Bacterial Meningitis ◽

Real Time ◽

Real Time Pcr ◽

Diagnostic Methods ◽

Type B ◽

E Coli ◽

Pcr Assays ◽

Set Up ◽

Etiological Agents ◽

Suis Serotype

Introduction: Bacterial meningitis is an acute central nervous infection with high mortality or permanent neurological sequelae if remained undiagnosed. However, traditional diagnostic methods for bacterial meningitis pose challenge in prompt and precise identification of causative agents. Aims: The present study will therefore aim to set up in-house PCR assays for diagnosis of six pathogens causing the disease including H. influenzae type b, S. pneumoniae, N. meningitidis, S. suis serotype 2, E. coli and S. aureus. Methods: inhouse PCR assays for detecting six above-mentioned bacteria were optimized after specific pairs of primers and probes collected from the reliable literature resources and then were performed for cerebrospinal fluid (CSF) samples from patients with suspected meningitis in Hue Hospitals. Results: The set of four PCR assays was developed including a multiplex real-time PCR for S. suis serotype 2, H. influenzae type b and N. meningitides; three monoplex real-time PCRs for E. coli, S. aureus and S. pneumoniae. Application of the in-house PCRs for 116 CSF samples, the results indicated that 48 (39.7%) cases were positive with S. suis serotype 2; one case was positive with H. influenzae type b; 4 cases were positive with E. coli; pneumococcal meningitis were 19 (16.4%) cases, meningitis with S. aureus and N. meningitidis were not observed in any CSF samples in this study. Conclusion: our in-house real-time PCR assays are rapid, sensitive and specific tools for routine diagnosis to detect six mentioned above meningitis etiological agents. Key words: Bacterial meningitis, etiological agents, multiplex real-time PCR

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Role of Laboratory Medicine in SARS-CoV-2 Diagnostics. Lessons Learned from a Pandemic

Healthcare ◽

10.3390/healthcare9070915 ◽

2021 ◽

Vol 9 (7) ◽

pp. 915

Author(s):

Irena Duś-Ilnicka ◽

Aleksander Szymczak ◽

Małgorzata Małodobra-Mazur ◽

Miron Tokarski

Keyword(s):

Molecular Biology ◽

Real Time ◽

Real Time Pcr ◽

Laboratory Medicine ◽

Lessons Learned ◽

Diagnostic Methods ◽

Medical Community ◽

Medical Diagnostic ◽

Novel Coronavirus ◽

Available Information

Since the 2019 novel coronavirus outbreak began in Wuhan, China, diagnostic methods in the field of molecular biology have been developing faster than ever under the vigilant eye of world’s research community. Unfortunately, the medical community was not prepared for testing such large volumes or ranges of biological materials, whether blood samples for antibody immunological testing, or salivary/swab samples for real-time PCR. For this reason, many medical diagnostic laboratories have made the switch to working in the field of molecular biology, and research undertaken to speed up the flow of samples through laboratory. The aim of this narrative review is to evaluate the current literature on laboratory techniques for the diagnosis of SARS-CoV-2 infection available on pubmed.gov, Google Scholar, and according to the writers’ knowledge and experience of the laboratory medicine. It assesses the available information in the field of molecular biology by comparing real-time PCR, LAMP technique, RNA sequencing, and immunological diagnostics, and examines the newest techniques along with their limitations for use in SARS-CoV-2 diagnostics.

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Detection of transgenic rice line TT51-1 in processed foods using conventional PCR, real-time PCR, and droplet digital PCR

Food Control ◽

10.1016/j.foodcont.2018.11.032 ◽

2019 ◽

Vol 98 ◽

pp. 380-388 ◽

Cited By ~ 3

Author(s):

Xiaofu Wang ◽

Ting Tang ◽

Qingmei Miao ◽

Shilong Xie ◽

Xiaoyun Chen ◽

...

Keyword(s):

Real Time ◽

Transgenic Rice ◽

Real Time Pcr ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Processed Foods ◽

Conventional Pcr ◽

Rice Line ◽

Transgenic Rice Line

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Cryptosporidium spp. during chemotherapy: a cross-sectional study of 94 patients with malignant solid tumor

Annals of Saudi Medicine ◽

10.5144/0256-4947.2021.293 ◽

2021 ◽

Vol 41 (5) ◽

pp. 293-298

Author(s):

Mehmet Karabey ◽

Hüseyin Can ◽

Tülay Öncü Öner ◽

Mert Döşkaya ◽

Sedef Erkunt Alak ◽

...

Keyword(s):

Sample Size ◽

Real Time ◽

Solid Tumors ◽

Real Time Pcr ◽

Tertiary Care ◽

Diagnostic Methods ◽

Cross Sectional ◽

Cryptosporidium Spp ◽

Stool Samples ◽

Pcr Targeting

BACKGROUND: Cryptosporidium spp . is a protozoan parasite that infects many vertebrate animals, including humans. Since Cryptosporidium spp . can cause chronic life-threatening diarrhea and severe malabsorption in immunocompromised patients, we investigated the prevalence of this parasite among patients undergoing chemotherapy for malignant solid tumors. OBJECTIVE: Investigate the prevalence of Cryptosporidium spp . in stool samples. DESIGN: Cross-sectional. SETTING: Tertiary care. PATIENTS AND METHODS: Stool samples were collected from adult patients with malignant solid tumors receiving chemotherapy and diarrhea. Cryptosporidium spp . prevalence was determined using Ziehl–Neelsen staining, ELISA, and real-time PCR targeting of the COWP gene. MAIN OUTCOME MEASURE: The prevalence of Cryptosporidium spp . in patients undergoing chemotherapy for malignant solid tumors. SAMPLE SIZE: 94 RESULTS: The prevalence was 2.1% (2/94), 5.3% (5/94), and 5.3% (5/94) as detected by Ziehl–Neelsen staining, real-time PCR and ELISA, respectively. The prevalence reached 8.5% (8/94) using all results obtained from the three methods. Among eight positive stool samples, four were positive by at least two different methods (Ziehl–Neelsen staining-ELISA or ELISA-real-time PCR) whereas the remaining four were positive by either ELISA or real-time PCR. CONCLUSION: These findings show the risk of cryptosporidiosis in cancer patients and the necessity to use at least two diagnostic methods during the diagnosis of cryptosporidiosis to reach more accurate and trustworthy results. LIMITATIONS: Further studies with a larger sample size are recommended. CONFLICT OF INTEREST: None.

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A Set of Conventional and Multiplex Real-Time PCR Assays for Direct Detection of Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis in Citrus Fruits

Plant Disease ◽

10.1094/pdis-05-18-0798-re ◽

2019 ◽

Vol 103 (2) ◽

pp. 345-356 ◽

Cited By ~ 4

Author(s):

Yosra Ahmed ◽

Jacqueline Hubert ◽

Céline Fourrier-Jeandel ◽

Megan M. Dewdney ◽

Jaime Aguayo ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Culture Media ◽

Direct Detection ◽

Elongation Factor ◽

Single Copy ◽

The United States ◽

Performance Criteria ◽

In Planta ◽

Conventional Pcr

Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis are causal agents of citrus scab and spot diseases. The three pathogens are listed as quarantine pests in many countries and are subject to phytosanitary measures to prevent their entry. Diagnosis of these diseases based on visual symptoms is problematic, as they could be confused with other citrus diseases. Isolation of E. fawcettii, E. australis, and P. angolensis from infected tissues is challenging because they grow slowly on culture media. This study developed rapid and specific detection tools for the in planta detection of these pathogens, using either conventional PCR or one-tube multiplex real-time PCR. Primers and hybridization probes were designed to target the single-copy protein-coding gene MS204 for E. fawcettii and E. australis and the translation elongation factor (Tef-1α) gene for P. angolensis. The specificity of the assays was evaluated by testing against DNA extracted from a large number of isolates (102) collected from different citrus-growing areas in the world and from other hosts. The newly described species E. citricola was not included in the specificity test due to its unavailability from the CBS collection. The detection limits of conventional PCR for the three pathogens were 100, 100, and 10 pg μl−1 gDNA per reaction for E. fawcettii, E. australis, and P. angolensis, respectively. The quadruplex qPCR was fully validated assessing the following performance criteria: sensitivity, specificity, repeatability, reproducibility, and robustness. The quadruplex real-time PCR proved to be highly sensitive, detecting as low as 243, 241, and 242 plasmidic copies (pc) μl−1 of E. fawcettii, E. australis, and P. angolensis, respectively. Sensitivity and specificity of this quadruplex assay were further confirmed using 176 naturally infected citrus samples collected from Ethiopia, Cameroon, the United States, and Australia. The quadruplex assay developed in this study is robust, cost-effective, and capable of high-throughput detection of the three targets directly from citrus samples. This new detection tool will substantially reduce the turnaround time for reliable species identification and allow rapid response and appropriate action.

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Development and application of a triplex real-time PCR assay for simultaneous detection of avian influenza virus, Newcastle disease virus, and duck Tembusu virus

BMC Veterinary Research ◽

10.1186/s12917-020-02399-z ◽

2020 ◽

Vol 16 (1) ◽

Author(s):

Xiyu Zhang ◽

Ming Yao ◽

Zhihui Tang ◽

Daning Xu ◽

Yan Luo ◽

...

Keyword(s):

Influenza Virus ◽

Standard Deviation ◽

Real Time ◽

Real Time Pcr ◽

Virus Isolation ◽

Simultaneous Detection ◽

Conventional Pcr ◽

Pcr Assay ◽

Duck Tembusu Virus ◽

Relative Standard

Abstract Background Pathogens including duck-origin avian influenza virus (AIV), duck-origin Newcastle disease virus (NDV) and duck Tembusu virus (DTMUV) posed great harm to ducks and caused great economic losses to the duck industry. In this study, we aim to develop a triplex real-time polymerase chain reaction (PCR) assay to detect these three viruses as early as possible in the suspicious duck flocks. Results The detection limit of the triplex real-time PCR for AIV, NDV, and DTMUV was 1 × 101 copies/μL, which was at least 10 times higher than the conventional PCR. In addition, the triplex assay was highly specific, and won’t cross-react with other duck pathogens. Besides, the intra-day relative standard deviation and inter-day relative standard deviation were lower than 4.44% for these viruses at three different concentrations. Finally, a total of 120 clinical samples were evaluated by the triplex real-time PCR, the conventional PCR and virus isolation, and the positive rates for these three methods were 20.83, 21.67, 19.17%, respectively. Taking virus isolation as the gold standard, the diagnostic specificity and positive predictive value of the three viruses were all above 85%, while the diagnostic sensitivity and negative predictive value of the three viruses were all 100%. Conclusion The developed triplex real-time PCR is fast, specific and sensitive, and is feasible and effective for the simultaneous detection of AIV, NDV, and DTMUV in ducks.

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