scholarly journals An Editing-Site-Specific PCR Method for Detection and Quantification of CAO1-Edited Rice

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1209
Author(s):  
Hongwen Zhang ◽  
Jun Li ◽  
Shengbo Zhao ◽  
Xiaohong Yan ◽  
Nengwu Si ◽  
...  
Keyword(s):  
Genetically Modified Organisms ◽  
High Specificity ◽  
Editing Site ◽  
Locked Nucleic Acid ◽  
Molecular Characteristics ◽  
Site Specific ◽  
Specific Pcr ◽  
Specificity And Sensitivity ◽  
Pcr Method ◽  
Detection And Quantification

Genome-edited plants created by genome editing technology have been approved for commercialization. Due to molecular characteristics that differ from classic genetically modified organisms (GMOs), establishing regulation-compliant analytical methods for identification and quantification of genome-edited plants has always been regarded as a challenging task. An editing-site-specific PCR method was developed based on the unique edited sequence in CAO1-edited rice plants. Test results of seven primer/probe sets indicated that this method can identify specific CAO1-edited rice from other CAO1-edited rice and wild types of rice with high specificity and sensitivity. The use of LNA (locked nucleic acid) in a probe can efficiently increase the specificity of the editing-site-specific PCR method at increased annealing temperature which can eliminate non-specific amplification of the non-target. The genome-edited ingredient content in blinded samples at the level of 0.1% to 5.0% was accurately quantified by this method on the ddPCR platform with RSD of <15% and bias in the range of ±17%, meeting the performance requirements for GMO detection method. The developed editing-site-specific PCR method presents a promising detection and quantification technique for genome-edited plants with known edited sequence.

scholarly journals Development of a triplex real-time PCR for simultaneous detection of allergenic ingredients in processed food

2018 ◽  
Vol 36 (No. 1) ◽  
pp. 22-27 ◽  
Author(s):  
Wenju Zhang ◽  
Yulei Zhao ◽  
Qingjin Xu ◽  
Qin Chen
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
High Specificity ◽  
Processed Food ◽  
Specific Pcr ◽  
Specificity And Sensitivity ◽  
Pcr Method ◽  
Primer Sets ◽  
Specific Pcr Primer ◽  
Suitable System

SYBR Green real-time or quantitative PCR (Q-PCR) is a suitable system in which to establish a multiplex method to detect allergenic ingredients in food. In this study, a triplex Q-PCR method was developed to detect trace amounts of peanut, soybean and sesame in processed food. Specific PCR primer sets were designed and the concentration of the primers used in the triplex PCR was optimised. The triplex method showed high specificity and sensitivity which were similar to those of the simplex method, and it was applied for the detection of allergenic ingredients in commercially available processed food. The results demonstrate that the developed triplex Q-PCR is a quick, reliable and efficient method for the detection of allergenic ingredients in processed food.

scholarly journals An alternative PCR assay with high sensitivity and specificity for the detection of swine toxoplasmosis based on the GRA14 gene

2021 ◽  
Author(s):  
Xi He ◽  
Derong Zhou ◽  
Yanwu Sun ◽  
Yuan Zhang ◽  
Xiaogang Zhang ◽  
...  
Keyword(s):  
Detection Method ◽  
Southern China ◽  
Acute Infection ◽  
High Sensitivity ◽  
High Specificity ◽  
Pcr Detection ◽  
Pcr Assay ◽  
Specific Primers ◽  
Specificity And Sensitivity ◽  
Pcr Method

Abstract Background Toxoplasma gondii, an intracellular apicomplexan protozoan parasite, can infect all warm-blooded animals. Infected swine are considered one of the most important sources of T. gondii infection in humans. Rapidly and effectively diagnosing T. gondii infection in swine is essential. PCR-based diagnostic tests have been fully developed, and very sensitive and specific PCR is crucial for the diagnosis of swine toxoplasmosis. Methods To established a high specificity and sensitivity PCR detection method for swine toxoplasmosis, we used T. gondii GRA14 gene as target to design specific primers and established a PCR detection method for swine toxoplasmosis. A total of 5462 blood specimens collected from pigs in 5 provinces and autonomous regions in southern China during 2016–2017 were assessed by the newly established GRA14 gene PCR method. Result Altogether, we used T. gondii GRA14 gene as target to design specific primers and established a high specificity and sensitivity PCR detection method for swine toxoplasmosis; in particular, this PCR method could detect T. gondii tachyzoite DNA in the acute infection phase. The GRA14 gene PCR assay detected a minimum of 2.35 tachyzoites of T. gondii, and it could be used for T. gondii detection in blood, tissue, semen, urine and waste feed specimens. The overall T. gondii infection rate was 18.9% (1033/5462) by the newly established GRA14 gene PCR method. According to statistical analysis among different regions, the positive rates of swine toxoplasmosis in the Shaanxi, Fujian and Guangdong areas in China from 2016 to 2017 were the highest, at 31.7% (44/139), 21.9% (86/391) and 18.8% (874/4645), respectively (χ2 = 84.2, P < 0.0001). Specimens collected in 2017 had a higher positive rate (19.1% or 886/4639) than those collected in 2016 (16.1% or 155/963) (χ2 = 4.5, P < 0.05). Specimens collected in autumn (39.4% or 187/474), spring (22.8% or 670/2940) and winter (18.2% or 129/709) also had higher positive rates than those collected in summer (3.8% or 57/1479) (χ2 = 427.7, P < 0.0001). Conclusions These results indicate that the new PCR method based on the T. gondii GRA14 gene would be useful for the diagnosis of swine toxoplasmosis and that it would facilitate the diagnosis of toxoplasmosis in clinical laboratories.

scholarly journals Restriction-Site-Specific PCR as a Rapid Test To Detect Enterohemorrhagic Escherichia coli O157:H7 Strains in Environmental Samples

2000 ◽  
Vol 66 (6) ◽  
pp. 2513-2519 ◽  
Author(s):  
Richard Kimura ◽  
Robert E. Mandrell ◽  
John C. Galland ◽  
Doreene Hyatt ◽  
Lee W. Riley
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibody ◽  
Polyclonal Antibody ◽  
Environmental Samples ◽  
Restriction Site ◽  
Site Specific ◽  
E Coli ◽  
Specific Pcr ◽  
Antibody Elisa ◽  
Pcr Method

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen in industrialized countries. We developed a rapid and simple test for detecting E. coli O157:H7 using a method based on restriction site polymorphisms. Restriction-site-specific PCR (RSS-PCR) involves the amplification of DNA fragments using primers based on specific restriction enzyme recognition sequences, without the use of endonucleases, to generate a set of amplicons that yield “fingerprint” patterns when resolved electrophoretically on an agarose gel. The method was evaluated in a blinded study of E. coli isolates obtained from environmental samples collected at beef cattle feedyards. The 54 isolates were all initially identified by a commonly used polyclonal antibody test as belonging to O157:H7 serotype. They were retested by anti-O157 and anti-H7 monoclonal antibody enzyme-linked immunosorbent assay (ELISA). The RSS-PCR method identified all 28 isolates that were shown to be E. coli O157:H7 by the monoclonal antibody ELISA as belonging to the O157:H7 serotype. Of the remaining 26 ELISA-confirmed non-O157:H7 strains, the method classified 25 strains as non-O157:H7. The specificity of the RSS-PCR results correlated better with the monoclonal antibody ELISA than with the polyclonal antibody latex agglutination tests. The RSS-PCR method may be a useful test to distinguish E. coli O157:H7 from a large number ofE. coli isolates from environmental samples.

FnCas12a/crRNA assisted dumbbell-PCR detection of IsomiRs with terminal and inner sequence variants

2020 ◽  
Vol 56 (69) ◽  
pp. 10038-10041 ◽  
Author(s):  
Junyan Wang ◽  
Tao Bing ◽  
Nan Zhang ◽  
Xiangjun Liu ◽  
Dihua Shangguan
Keyword(s):  
High Specificity ◽  
Pcr Detection ◽  
Sequence Variants ◽  
Specificity And Sensitivity ◽  
Pcr Method

A new FnCas12a/crRNA assisted Dumbbell-PCR (Cas-Db-PCR) method is developed for the detection of isomiR heterogeneity in length and/or sequence variants with high specificity and sensitivity.

Real-Time PCR Method for Quantification of Staphylococcus aureus in Milk

2007 ◽  
Vol 70 (1) ◽  
pp. 90-96 ◽  
Author(s):  
M. GOTO ◽  
H. TAKAHASHI ◽  
Y. SEGAWA ◽  
H. HAYASHIDANI ◽  
K. TAKATORI ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Real Time ◽  
Real Time Pcr ◽  
Pure Culture ◽  
High Specificity ◽  
Pcr Assay ◽  
Milk Samples ◽  
High Temperature Short Time ◽  
Specificity And Sensitivity ◽  
Pcr Method

A reproducible real-time PCR method that targets the putative transcriptional regulator gene of Staphylococcus aureus was developed to quantify this microorganism in milk samples. On the basis of partial sequences of this gene determined from S. aureus strains, we designed the specific primers and probe for use in a quantitative PCR assay. These specificities were confirmed with 25 strains of S. aureus and 35 strains of other bacteria. A real-time PCR assay with serial 10-fold dilutions of purified DNA and pure culture was conducted. It was possible to construct standard curves with a high correlation coefficient (r2 = 0.99) in the range of 50 ng to 50 fg for purified DNA and 107 to 101 CFU/ml for a pure culture. The constructed standard curve for milk samples was similar to that for the pure culture, and the quantification of S. aureus in the range of 107 to 101 CFU/ml was possible. Moreover, to determine how our real-time PCR method would perform under actual analytical conditions, we quantified the DNA from S. aureus after two types of heat treatments were used for the pasteurization of milk. The amount of DNA found was affected after heat treatment at 63°C for 30 min (low-temperature long-time method) but not at 72°C for 15 s (high-temperature short-time method). The results indicate that the real-time PCR method developed in this study is effective for monitoring S. aureus contamination in milk because of its high specificity and sensitivity.

scholarly journals Rapid Detection of Pine Pathogens Lecanosticta acicola, Dothistroma pini and D. septosporum on Needles by Probe-Based LAMP Assays

Forests ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 479
Author(s):  
Chiara Aglietti ◽  
Colton D. Meinecke ◽  
Luisa Ghelardini ◽  
Irene Barnes ◽  
Ariska van der Nest ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Morphological Characteristics ◽  
Lamp Assay ◽  
High Specificity ◽  
Portable Devices ◽  
Forest Protection ◽  
Specific Pcr ◽  
Specificity And Sensitivity ◽  
Lecanosticta Acicola ◽  
Dothistroma Pini

Needle blights are serious needle fungal diseases affecting pines both in natural and productive forests. Among needle blight agents, the ascomycetes Lecanosticta acicola, Dothistroma pini and D. septosporum are of particular concern. These pathogens need specific, fast and accurate diagnostics since they are regulated species in many countries and may require differential management measures. Due to the similarities in fungal morphology and the symptoms they elicit, these species are hard to distinguish using morphological characteristics. The symptoms can also be confused with those caused by insects or abiotic agents. DNA-based detection is therefore recommended. However, the specific PCR assays that have been produced to date for the differential diagnosis of these pathogens can be applied only in a well-furnished laboratory and the procedure takes a relatively long execution time. Surveillance and forest protection would benefit from a faster diagnostic method, such as a loop-mediated isothermal amplification (LAMP) assay, which requires less sophisticated equipment and can also be deployed directly on-site using portable devices. LAMP assays for the rapid and early detection of L. acicola, D. pini and D. septosporum were developed in this work. Species-specific LAMP primers and fluorescent assimilating probes were designed for each assay, targeting the beta tubulin (β-tub2) gene for the two Dothistroma species and the elongation factor (EF-1α) region for L. acicola. Each reaction detected its respective pathogen rapidly and with high specificity and sensitivity in DNA extracts from both pure fungal cultures and directly from infected pine needles. These qualities and the compatibility with inexpensive portable instrumentation position these LAMP assays as an effective method for routine phytosanitary control of plant material in real time, and they could profitably assist the management of L. acicola, D. pini and D. septosporum.

scholarly journals Application of PCR-ELISA in Molecular Diagnosis

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Mei Jean Sue ◽  
Swee Keong Yeap ◽  
Abdul Rahman Omar ◽  
Sheau Wei Tan
Keyword(s):  
Detection Limit ◽  
High Specificity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specificity And Sensitivity ◽  
Pcr Product ◽  
Diagnostic Time ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Agricultural Industries ◽  
Analytical Time

Polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) is an immunodetection method that can quantify PCR product directly after immobilization of biotinylated DNA on a microplate. This method, which detects nucleic acid instead of protein, is a much more sensitive method compared to conventional PCR method, with shorter analytical time and lower detection limit. Its high specificity and sensitivity, together with its semiquantitative ability, give it a huge potential to serve as a powerful detection tool in various industries such as medical, veterinary, and agricultural industries. With the recent advances in PCR-ELISA, it is envisaged that the assay is more widely recognized for its fast and sensitive detection limit which could improve overall diagnostic time and quality.

scholarly journals Detection of Legionella Species in Respiratory Specimens Using PCR with Sequencing Confirmation

2000 ◽  
Vol 38 (5) ◽  
pp. 1709-1712 ◽  
Author(s):  
J. L. Cloud ◽  
K. C. Carroll ◽  
P. Pixton ◽  
M. Erali ◽  
D. R. Hillyard
Keyword(s):  
Legionella Pneumophila ◽  
High Sensitivity ◽  
High Specificity ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Pcr Method ◽  
Specific Pcr Assay ◽  
Clinically Significant ◽  
Tract Infections ◽  
Culture Negative

Legionella spp. are a common cause of community-acquired respiratory tract infections and an occasional cause of nosocomial pneumonia. A PCR method for the detection of legionellae in respiratory samples was evaluated and was compared to culture. The procedure can be performed in 6 to 8 h with a commercially available DNA extraction kit (Qiagen, Valencia, Calif.) and by PCR with gel detection. PCR is performed with primers previously determined to amplify a 386-bp product within the 16S rRNA gene of Legionella pneumophila. We can specifically detect the clinically significant Legionella species including L. pneumophila, L. micdadei, L. longbeachae, L. bozemanii, L. feeleii, and L. dumoffii. The assay detects 10 fg (approximately two organisms) of legionella DNA in each PCR. Of 212 clinical specimens examined by culture, 100% of the culture-positive samples (31 of 31) were positive by this assay. By gel detection of amplification products, 12 of 181 culture-negative samples were positive forLegionella species by PCR, resulting in 93% specificity. Four of the 12 samples with discrepant results (culture negative, PCR positive) were confirmed to be positive for Legionellaspecies by sequencing of the amplicons. The legionella-specific PCR assay that is described demonstrates high sensitivity and high specificity for routine detection of legionellae in respiratory samples.

scholarly journals An Optimized Real-Time PCR to Avoid Species-/Tissue-Associated Inhibition for H5N1 Detection in Ferret and Monkey Tissues

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
LingJun Zhan ◽  
LinLin Bao ◽  
FengDi Li ◽  
Qi Lv ◽  
LiLi Xu ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rna Isolation ◽  
High Specificity ◽  
Sybr Green ◽  
Pcr Inhibitors ◽  
Specificity And Sensitivity ◽  
Avian Influenza Virus H5n1 ◽  
Pcr Method ◽  
Tissue Specimens

The real-time PCR diagnostics for avian influenza virus H5N1 in tissue specimens are often suboptimal, since naturally occurring PCR inhibitors present in samples, or unanticipated match of primer to unsequenced species’ genome. With the principal aim of optimizing the SYBR Green real-time PCR method for detecting H5N1 in ferret and monkey (Chinese rhesus macaque) tissue specimens, we screened various H5N1 gene-specific primer pairs and tested their ability to sensitively and specifically detect H5N1 transcripts in the infected animal tissues, then we assessed RNA yield and quality by comparing Ct values obtained from the standard Trizol method, and four commonly used RNA isolation kits with small modifications, including Roche High Pure, Ambion RNAqueous, BioMIGA EZgene, and Qiagen RNeasy. The results indicated that a single primer pair exhibited high specificity and sensitivity for H5N1 transcripts in ferret and monkey tissues. Each of the four kits and Trizol reagent produced high-quality RNA and removed all or nearly all PCR inhibitors. No statistically significant differences were found between the Ct values from the isolation methods. So the optimized SYBR Green real-time PCR could avoid species- or tissue-associated PCR inhibition in detecting H5N1 in ferret and monkey tissues, including lung and small intestine.

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