Direct detection of Mycoplasma bovis in milk and tissue samples by real-time PCR

Abstract objective: Histopathological studies suggest that parasite load is different between acute and chronic forms of cutaneous leishmaniasis (CL). However, highly sensitive detection methods are still needed to distinguish different forms of leishmaniasis. In the present study, we developed a quantitative real-time polymerase chain reaction (PCR) to detect and quantify leishmania tropica parasites in paraffin-embedded tissue samples. Results: The ability of real-time PCR for leishmania detection was higher than histopathological evaluation. The parasite loads were quantified by qPCR assay and microscopic evaluation were highly correlated ( r =0.598; P <0.001). Among patients, the parasite load was inversely correlated with disease duration (acute CL lesions had very higher parasite loads than chronic CL lesions), but there was no difference in parasite load according to the patients’ age and sex as well as location of the lesions. In contrast to Ridley scoring system (P<0.001), there were no statistically significant differences in the relative number of parasites among the lupoid and non-lupoid forms of chronic lesions in real-time PCR (P=0.549), which indicates the superiority of histopathological evaluation in CL forms differentiation.

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Direct detection of microorganisms responsible for the sexually transmitted infection on the mobile real-time PCR device without treating in advance

10.1101/2021.03.02.21252726 ◽

2021 ◽

Author(s):

Masaaki Muraoka ◽

Kazunori Sohma ◽

Osamu Kawaguchi ◽

Mikio Mizukoshi

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Direct Detection ◽

Treponema Pallidum ◽

Nucleic Acid Amplification ◽

Direct Pcr ◽

Transmitted Infection ◽

Sexually Transmitted ◽

Ct Value

ABSTRACTAs WHO reported, four curable STIs-chlamydia, gonorrhoea, syphilis and trichomoniasis occur more than 1 million per each day globally almond 2016. For this reason, it is important to control these STIs, one of which is “to detect”. The general methods in order to detect STIs are nucleic acid amplification tests (NAATs). One of the reasons why NAATs are utilized in many tests is that it is possibly to be more sensitive than other test. However, there needs to treat extraction of nucleic acids in advance and amplify specific regions by NAATs, and hence it must take much labour and much time. In this work, for Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Treponema pallidum (TP) which is each etiological agent of chlamydia, gonorrhoea and syphilis, we evaluate and propose “quicker and simpler” NAATs. Specifically, utilizing mobile real-time PCR device “PCR1100” and PCR reagent kit “KAPA3G Plant PCR Kit”, it was considered whether real-time direct PCR could be performed or not without treating DNA extraction in advance so-called “direct”.As a result, firstly, we established that real-time direct PCR could be performed in all of CT, NG, and TP, and moreover, each Ct value correlated with the concentration of each organism similarly to detection of genome DNA (each correlation coefficient R2 > 0.95). Moreover, each assay demonstrated a limit of detection (LOD) of the follows; CT was 10^0.86 = 7.24 IFU/reaction, NG was 10^-0.19 = 0.65 CFU/reaction, and TP was 10^1.4 = 25.1 organisms/reaction. However, it appeared the sensitivity was a little low, especially for CT and TP.Secondly, we found that even as without treating sample in advance, the time of detection was required more less 15 minutes at any of case, which was very quick compared with other current methods for real-time PCR. Additionally, compared with other commercial devices, it was easier to operate the PCR1100 device, for example, start, analysis of Ct value.In conclusion, the present study has demonstrated that it is possible for real-time direct PCR to perform with combination of the PCR1100 device and the PCR reagent kit in 3 kinds of microorganisms-CT, NG and TP. Furthermore, we propose “quicker and simpler” methods for NAATs, which it would not take labour and time. Further studies are needed in order to contribute to control STIs.

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Detection of Jaagsiekte sheep retrovirus in apparently healthy sheep by real-time TaqMan PCR in comparison with histopathological findings

Journal of Veterinary Research ◽

10.1515/jvetres-2016-0002 ◽

2016 ◽

Vol 60 (1) ◽

pp. 7-12 ◽

Cited By ~ 2

Author(s):

Aliasghar Bahari ◽

Masoud Sabouri Ghannad ◽

Omid Dezfoulian ◽

Fereydon Rezazadeh ◽

Ali Sadeghi-Nasab

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Mediastinal Lymph Node ◽

Pulmonary Adenocarcinoma ◽

Ovine Pulmonary Adenocarcinoma ◽

Jaagsiekte Sheep Retrovirus ◽

Tissue Samples ◽

Blood Samples ◽

Proviral Dna ◽

Healthy Sheep

Abstract Introduction: The aim of this study was to use TaqMan real-time PCR technique to investigate Jaagsiekte sheep retrovirus (JSRV) proviral DNA in whole blood samples of sheep, and compare the results to those of histopathological examinations. Material and Methods: Eighty blood samples from clinically healthy sheep were randomly collected before the animals were slaughtered. Ten tissue samples from each lung and associated caudal mediastinal lymph node were taken. Results: Fifteen (18.75%) blood samples were found to contain proviral DNA, and 11 (13.75%) corresponding lung samples showed microscopic changes consistent with ovine pulmonary adenocarcinoma. None of the samples displayed metastases to the caudal mediastinal lymph nodes. The prominent pattern of neoplastic nodules consisted of acinar (alveolar) form. Conclusion: The results indicated the higher sensitivity of real-time PCR compared to histopathological examinations in detection of ovine pulmonary adenocarcinoma.

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Direct detection ofCampylobacter jejuniin human stool samples by real-time PCR

Canadian Journal of Microbiology ◽

10.1139/w08-064 ◽

2008 ◽

Vol 54 (9) ◽

pp. 742-747 ◽

Cited By ~ 11

Author(s):

Shiyong Lin ◽

Xinying Wang ◽

Haoxuan Zheng ◽

Zhengguo Mao ◽

Yong Sun ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Direct Detection ◽

Culture Method ◽

Turnaround Time ◽

Culture Methods ◽

Time Saving ◽

Whole Process ◽

Pure Cultures ◽

Stool Samples

Our purpose was to establish a quick and accurate real-time PCR (rtPCR) method to detect Campylobacter jejuni directly from human diarrheal stool as an alternative to traditional culture methods. To determine the consistency of rtPCR and culture method, 256 clinical diarrheal stool samples and 50 normal stool samples from healthy individuals were examined, and the whole process was double-blinded. Our data showed that the sensitivity of rtPCR in pure cultures and stool was 102CFU·mL–1and 103CFU·g–1, respectively. Of the 256 diarrheal samples, 10 specimens were successfully detected by both methods, whereas two specimens were PCR positive but culture negative. No positive results were found by these two methods in 50 normal specimens. Our data suggested that rtPCR was convenient in operation and time-saving (turnaround time 3.5–4 h), so it could be used for clinical diagnostic and epidemiological purposes.

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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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Real-time PCR assay for detecting illicit steroid administration in veal calves allows reliable biomarker profiling of formalin-fixed, paraffin-embedded (FFPE) archival tissue samples

Food Chemistry ◽

10.1016/j.foodchem.2019.126061 ◽

2020 ◽

Vol 312 ◽

pp. 126061

Author(s):

Alessandro Benedetto ◽

Marzia Pezzolato ◽

Chiara Beltramo ◽

Valentina Audino ◽

Francesco Ingravalle ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Assay ◽

Tissue Samples ◽

Archival Tissue ◽

Steroid Administration ◽

Veal Calves ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

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Examination of Mycobacterium avium subsp. avium distribution in naturally infected hens by culture and triplex quantitative real time PCR

Veterinární Medicína ◽

10.17221/2966-vetmed ◽

2010 ◽

Vol 55 (No. 7) ◽

pp. 325-330 ◽

Cited By ~ 4

Author(s):

M. Kaevska ◽

I. Slana ◽

P. Kralik ◽

I. Pavlik

Keyword(s):

Real Time ◽

Mycobacterium Avium ◽

Real Time Pcr ◽

Clinical Symptoms ◽

Bird Species ◽

Etiologic Agent ◽

Mycobacterium Avium Subsp ◽

Quantitative Real Time Pcr ◽

Tissue Samples ◽

Conventional Culture

Mycobacterium avium subsp. avium (MAA) is the etiologic agent of avian tuberculosis, a chronic contagious disease described in a wide variety of domestic and wild bird species. The aims of this study were to assess the advantages of triplex quantitative real time PCR (qPCR) in comparison with culture testing for distribution of MAA in the organs of hens displaying varying degrees of clinical symptoms of the disease. From one small flock of ten hens and one cock with a history of weight loss, 98 tissue samples were examined in total. Pathological lesions were observed in six hens from which two were clinically ill. A total of 12 samples were positive by culture and 16 were positive by IS901 and IS1245 qPCR, confirming MAA infection. In conclusion, qPCR was a faster and more reliable alternative method in comparison with conventional culture analysis. Due to the detection of MAA in the muscle tissue of one hen, consumption of under cooked meat originating from infected fowl could pose a threat to immunosuppressed individuals.

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Detection and Enumeration of Salmonella Enteritidis in Homemade Ice Cream Associated with an Outbreak: Comparison of Conventional and Real-Time PCR Methods

Journal of Food Protection ◽

10.4315/0362-028x-69.3.639 ◽

2006 ◽

Vol 69 (3) ◽

pp. 639-643 ◽

Cited By ~ 27

Author(s):

K. H. SEO ◽

I. E. VALENTIN-BON ◽

R. E. BRACKETT

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Real Time Pcr ◽

Salmonella Enteritidis ◽

Direct Detection ◽

Ice Cream ◽

Plate Count ◽

Pcr Assay ◽

The Real ◽

Real Time Quantitative Pcr

Salmonellosis caused by Salmonella Enteritidis (SE) is a significant cause of foodborne illnesses in the United States. Consumption of undercooked eggs and egg-containing products has been the primary risk factor for the disease. The importance of the bacterial enumeration technique has been enormously stressed because of the quantitative risk analysis of SE in shell eggs. Traditional enumeration methods mainly depend on slow and tedious most-probable-number (MPN) methods. Therefore, specific, sensitive, and rapid methods for SE quantitation are needed to collect sufficient data for risk assessment and food safety policy development. We previously developed a real-time quantitative PCR assay for the direct detection and enumeration of SE and, in this study, applied it to naturally contaminated ice cream samples with and without enrichment. The detection limit of the real-time PCR assay was determined with artificially inoculated ice cream. When applied to the direct detection and quantification of SE in ice cream, the real-time PCR assay was as sensitive as the conventional plate count method in frequency of detection. However, populations of SE derived from real-time quantitative PCR were approximately 1 log higher than provided by MPN and CFU values obtained by conventional culture methods. The detection and enumeration of SE in naturally contaminated ice cream can be completed in 3 h by this real-time PCR method, whereas the cultural enrichment method requires 5 to 7 days. A commercial immunoassay for the specific detection of SE was also included in the study. The real-time PCR assay proved to be a valuable tool that may be useful to the food industry in monitoring its processes to improve product quality and safety.

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