Multiplex Detection of Viral DNAs in Blood by Colorimetrically Identifying Polymerase Chain Reaction Amplicons with Serial Invasive Reaction Assisted Gold Nanoparticle Probes Assembling

2020 ◽  
Vol 20 (10) ◽  
pp. 6140-6147
Author(s):  
Yi Ma ◽  
Chunyan Gong ◽  
Xiemin Qi ◽  
Bingjie Zou ◽  
Qinxin Song ◽  
...  
Keyword(s):  
Real Time ◽  
Gold Nanoparticle ◽  
Real Time Pcr ◽  
Pathogen Detection ◽  
Colorimetric Assay ◽  
Treponema Pallidum ◽  
Multiplex Detection ◽  
Clinical Practices ◽  
Resource Limited ◽  
Viral Dnas

Detection of blood-borne pathogenic viruses is essential for blood transfusion, and has great significance for epidemiology, as well as clinical practices. Common blood-borne viruses causing infectious diseases include Hepatitis B virus (HBV), Hepatitis C virus (HCV), Human immunodeficiency virus (HIV) and Treponema pallidum (TP). Therefore, multiplex detection of these viruses is more in the line with the needs of clinical testing. Although real-time PCR-based multiplex nucleic acid testing (NAT) was developed for pathogen detection, however, the requirement of multichannel realtime PCR machine increases the instrumental cost and is not suitable for use in resource-limited areas. In this study, we proposed a multiplex and colorimetric assay for detecting viral nucleic acids in blood by using serial invasive reaction assisted gold nanoparticle (AuNPs) probes assembling to identify multiplex PCR amplicons. As low as 2 copies per reaction of HIV and TP targets, and 20 copies per reaction of HBV and HCV targets can be detected. The results can be observed by naked eyes; thus, just a standard PCR machine is required. In addition, the hairpin probe and the AuNPs for signal read out are universal for all the targets, reducing the detection cost. About 20 DNA samples remaining after clinical HBV testing were successfully detected, and the results were consistent with that of commercially available real-time PCR based kit, indicating that this method has a potential for clinical applications.

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

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.

scholarly journals Identification of Field Tolerance and Resistance to Mal Secco Disease in a Citrus Germplasm Collection in Sicily

Agronomy ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1806
Author(s):  
Riccardo Russo ◽  
Marco Caruso ◽  
Carmen Arlotta ◽  
Angela Roberta Lo Piero ◽  
Elisabetta Nicolosi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pathogen Detection ◽  
Germplasm Collection ◽  
Research Centre ◽  
Molecular Screening ◽  
Sources Of Resistance ◽  
Strong Negative Correlation ◽  
Canopy Volume ◽  
Mal Secco

Mal secco is a tracheomycotic disease caused by the fungus Plenodomus tracheiphilus (Petri) Gruyter, Aveskamp, and Verkley that has caused severe damage and loss of yield in the citrus industry in the Mediterranean area, for 100 years. While the disease can affect different cultivated citrus species, lemon (C. × limon var. limon (L.) Burm. f.) and citron are the most susceptible. The identification of resistant or field-tolerant clones and hybrids is a major goal for lemon growers and breeders. To identify sources of resistance or tolerance to the disease, we performed a phenotypic survey on a lemon and lemon-like open-field germplasm planted at CREA (Research Centre for Olive, Fruit and Citrus Crops), Italy, in an area with high pathogen pressure. Phenotyping was performed visually, four times, for three consecutive years, on a total of 50 accessions, with two or three replicate trees per accession. Moreover, molecular screening based on real-time PCR was performed, for two consecutive years, on twigs, young leaves, and mature leaves of all plants, to detect the pathogen in the absence of clear symptoms. The accessions were categorized into seven groups based on the presence of visual symptoms, real-time PCR pathogen detection, and canopy volume. The results revealed sources of tolerance in lemon and citron hybrids. The molecular screening identified P. tracheiphilus in all lemon clones, with mean Ct values ranging from 17 to 39. The screening also identified P. tracheiphilus in clones without clear symptoms, indicating their ability to tolerate the disease. Moreover, a strong negative correlation was found between the Ct values in twigs and symptom severity (r = −0.72). This indicates that the DNA from twigs is the most appropriate for use in performing reliable phenotyping of mal secco susceptibility in adult plants. An autotetraploid lemon (Doppio Lentini) seems to be immune to the disease, under natural pressure, since P. tracheiphilus was not detected by real-time PCR and visual screening. Overall, the data obtained are a valuable resource for identifying both the most tolerant lemon varieties suitable for areas with high pathogen pressure and the best breeding parents for the introgression of resistance genes into lemon genotypes.

Real-Time Nucleic Acid–Based Detection Methods for Pathogenic Bacteria in Food

2004 ◽  
Vol 67 (4) ◽  
pp. 823-832 ◽  
Author(s):  
JOHN L. McKILLIP ◽  
MARYANNE DRAKE
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Food Industry ◽  
Pathogenic Bacteria ◽  
Pathogen Detection ◽  
Food Microbiology ◽  
Detection Methods ◽  
Target Sequence ◽  
Bacterial Enumeration

Quality assurance in the food industry in recent years has involved the acceptance and implementation of a variety of nucleic acid–based methods for rapid and sensitive detection of food-associated pathogenic bacteria. Techniques such as polymerase chain reaction have greatly expedited the process of pathogen detection and have in some cases replaced traditional methods for bacterial enumeration in food. Conventional PCR, albeit sensitive and specific under optimized conditions, obligates the user to employ agarose gel electrophoresis as the means for endpoint analysis following sample processing. For the last few years, a variety of real-time PCR chemistries and detection instruments have appeared on the market, and many of these lend themselves to applications in food microbiology. These approaches afford a user the ability to amplify DNA or RNA, as well as detect and confirm target sequence identity in a closed-tube format with the use of a variety of fluorophores, labeled probes, or both, without the need to run gels. Such real-time chemistries also offer greater sensitivity than traditional gel visualization and can be semiquantitative and multiplexed depending on the specific experimental objectives. This review emphasizes the current systems available for real-time PCR–based pathogen detection, the basic mechanisms and requirements for each, and the prospects for development over the next few years in the food industry.

Rapid real-time PCR assay for detecting Salmonella in raw and ready-to-eat meats

2008 ◽  
Vol 56 (4) ◽  
pp. 451-458 ◽  
Author(s):  
Jitu Patel ◽  
Arvind Bhagwat
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pathogen Detection ◽  
Molecular Beacon ◽  
False Negative ◽  
Detection Methods ◽  
Time Data ◽  
Pcr Assay ◽  
Serovar Typhimurium ◽  
Meat Samples

A real-time PCR assay was evaluated for the rapid detection (10 h) ofSalmonellain meats using molecular beacon probes available as a commercial kit (iQ-Check, Bio-Rad laboratories). Raw (chicken, pork) and ready-to-eat (RTE) meats were artificially contaminated withSalmonella entericaserovar Typhimurium at the estimated level of 2 to 4 cells per 25 g. After 8 h of pre-enrichment in buffered peptone water, a molecular beacon-based PCR assay was performed to detect contamination in raw and RTE meats. The sensitivity and accuracy of the assay were compared with the conventional USDA microbiological procedure. Comparative evaluation of the USDA procedure with the rapid PCR assay for meat samples (n = 63) revealed 1 false negative pork sample with the PCR assay. All uninoculated controls (n = 34) but one sample were negative by both the 10-h PCR assay and the USDA procedure. Developing rapid pathogen detection methods with shorter pre-enrichment times (8-h) and real-time data monitoring capabilities will benefit the industry in preventing recall of contaminated meats by stopping the contaminated products from being introduced into the marketplace.

Comparison of conventional culture with SeptiFast real-time PCR for microbial pathogen detection in clinical specimens other than blood

2011 ◽  
Vol 60 (12) ◽  
pp. 1774-1778 ◽  
Author(s):  
Antonella Mencacci ◽  
Christian Leli ◽  
Angela Cardaccia ◽  
Paolo Montagna ◽  
Amedeo Moretti ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pathogen Detection ◽  
Clinical Specimens ◽  
Microbial Pathogen ◽  
Conventional Culture

scholarly journals Usefulness of Multiplex Real-Time PCR for Simultaneous Pathogen Detection and Resistance Profiling of Staphylococcal Bacteremia

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Yousun Chung ◽  
Taek Soo Kim ◽  
Young Gi Min ◽  
Yun Ji Hong ◽  
Jeong Su Park ◽  
...  
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
Pathogen Detection ◽  
Methicillin Resistance ◽  
Blood Cultures ◽  
Pcr Assay ◽  
Accurate Identification ◽  
Essential Information ◽  
Methicillin Resistant

Staphylococci are the leading cause of nosocomial blood stream infections. Fast and accurate identification of staphylococci and confirmation of their methicillin resistance are crucial for immediate treatment with effective antibiotics. A multiplex real-time PCR assay that targetsmecA,femAspecific forS. aureus,femAspecific forS. epidermidis,16S rRNAfor universal bacteria, and16S rRNAspecific for staphylococci was developed and evaluated with 290 clinical blood culture samples containing Gram-positive cocci in clusters (GPCC). For the 262 blood cultures identified to the species level with the MicroScan WalkAway system (Siemens Healthcare Diagnostics, USA), the direct real-time PCR assay of positive blood cultures showed very good agreement for the categorization of staphylococci into methicillin-resistantS. aureus(MRSA), methicillin-susceptibleS. aureus(MSSA), methicillin-resistantS. epidermidis(MRSE), methicillin-susceptibleS. epidermidis(MSSE), methicillin-resistant non-S. epidermidisCoNS (MRCoNS), and methicillin-susceptible non-S. epidermidisCoNS (MSCoNS) (κ=0.9313). The direct multiplex real-time PCR assay of positive blood cultures containing GPCC can provide essential information at the critical point of infection with a turnaround time of no more than 4 h. Further studies should evaluate the clinical outcome of using this rapid real-time PCR assay in glycopeptide antibiotic therapy in clinical settings.

scholarly journals Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™

BMC Genomics ◽  
2007 ◽  
Vol 8 (1) ◽  
pp. 276 ◽  
Author(s):  
Ronald van Doorn ◽  
Marianna Szemes ◽  
Peter Bonants ◽  
George A Kowalchuk ◽  
Joana F Salles ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Plant Pathogens ◽  
Multiplex Detection

scholarly journals Development of a high-throughput real-time PCR system for detection of enzootic pathogens in pigs

2019 ◽  
Vol 32 (1) ◽  
pp. 51-64 ◽  
Author(s):  
Nicole B. Goecke ◽  
Charlotte K. Hjulsager ◽  
Jesper S. Krog ◽  
Kerstin Skovgaard ◽  
Lars E. Larsen
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Pathogen Detection ◽  
Detection System ◽  
Working Hours ◽  
Negative Influence ◽  
Optimal Choice ◽  
National Veterinary Institute ◽  
Wide Range

Respiratory and intestinal diseases in pigs can have significant negative influence on productivity and animal welfare. A wide range of real-time PCR (rtPCR) assays are used in our laboratory (National Veterinary Institute, Technical University of Denmark) for pathogen detection, and PCR analyses are performed on traditional rtPCR platforms in which a limited number of samples can be analyzed per day given limitations in equipment and personnel. To mitigate these restrictions, rtPCR assays have been optimized for the high-throughput rtPCR BioMark platform (Fluidigm). Using this platform, we developed a high-throughput detection system that can be used for simultaneous examination of 48 samples with detection specificity for 18 selected respiratory and enteric viral and bacterial pathogens of high importance to Danish pig production. The rtPCR assays were validated and optimized to run under the same reaction conditions using a BioMark 48.48 dynamic array (DA) integrated fluidic circuit chip, and the sensitivity and specificity were assessed by testing known positive samples. Performance of the 48.48DA was similar to traditional rtPCR analysis, and the specificity of the 48.48DA was high. Application of the high-throughput platform has resulted in a significant reduction in cost and working hours and has provided production herds with a new innovative service with the potential to facilitate the optimal choice of disease control strategies such as vaccination and treatment.

scholarly journals Detection of the Downy Mildew Pathogen on Seed of Basil Following Field Infection in Southern New Jersey

2018 ◽  
Vol 28 (5) ◽  
pp. 637-641 ◽  
Author(s):  
Christian A. Wyenandt ◽  
Lisa R. Maimone ◽  
Kathryn Homa ◽  
Angela M. Madeiras ◽  
Robert L. Wick ◽  
...  
Keyword(s):  
New Jersey ◽  
Real Time ◽  
Downy Mildew ◽  
Real Time Pcr ◽  
Pathogen Detection ◽  
Agricultural Research ◽  
Pcr Analysis ◽  
Breeding Lines ◽  
Seed Collection ◽  
Seed Harvest

Different basils (Ocimum sp.) and cultivars (28 in 2009 and 32 in 2010) were evaluated for susceptibility to basil downy mildew (Peronospora belbahrii) at the Rutgers Agricultural Research and Extension Center near Bridgeton in southern New Jersey. At the end of each growing season, seed was collected from individual plants and stored for potential downy mildew pathogen detection using real-time polymerase chain reaction (PCR) analysis. Most of the basil cultivars and breeding lines were showing symptoms of basil downy mildew infection at the time of seed collection before the first frost near the end of the production season. Symptoms of basil downy mildew were present on 25 of the 28 (89%) basil lines evaluated in 2009 and 26 of 32 (81%) basil lines tested in 2010 at the time of seed harvest, with sporulation evident on the abaxial surface of infected leaves. Real-time PCR analysis of seed collected from various infected plants detected P. belbahrii on seed of 14 of 25 (56%) basil lines tested in 2009 and 8 of 32 (25%) tested in 2010. Importantly, P. belbahrii was not only detected on seed of sweet basil (Ocimum basilicum) phenotypes but also on seed of ‘Spice’ basil (Ocimum americanum) in 2009 and ‘Sweet Dani Lemon Basil’ basil (Ocimum citriodorum), ‘Holy Red and Green’ basil [Ocimum tenuiflorum (form. sanctum)], ‘Lime’ basil (O. americanum), and again on ‘Spice’ basil in 2010 where no symptoms (i.e., no chlorosis or sporulation) were present on the leaves when seed were collected. This work demonstrates that basil seed, regardless of basil species and whether symptoms are visible on foliage of the basil plant or the plant is immune or resistant to downy mildew, can test positive for the presence of P. belbahrii using a real-time PCR assay following exposure of plants to the pathogen during the natural development of downy mildew under field conditions.

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