Novel SYBR Green Real Time PCR Method for Direct Quantification and Detection of Escherichia coli from Clinical Samples

Pathogenic Escherichia coli (E. coli) has been implicated in a wide range of disease causing infections. It is essential to generate a method for detecting and differentiating each pathotype of E. coli which is more quickly and efficiently by using less reagent. This study aimed to evaluate a SYBR Green multiplex real-time PCR method for detecting four types of pathogenic E. coli. Two of multiplex real-time PCR system, 6-plex and 3-plex, were set to detect six different virulence factors from ETEC, EPEC, EHEC, and EIEC and evaluate the melting curves and specificity compared to simplex method. The results showed that 3-plex rt-PCR method gave more reliable melting curves than 6-plex. The 3-plex rt-PCR also provided similar melting value (Tm) to simplex system. The results of this specificity assay supported the selection of 3-plex rt-PCR conditions for detection of pathogenic E. coli.

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Development and evaluation of duplex TaqMan real-time PCR assay for detection and differentiation of wide-type and MGF505-2R gene-deleted African swine fever viruses

10.21203/rs.3.rs-59710/v2 ◽

2020 ◽

Author(s):

zhenhua Guo ◽

Kunpeng Li ◽

Songlin Qiao ◽

Xinxin Chen ◽

Ruiguang Deng ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Large Scale ◽

Limit Of Detection ◽

African Swine Fever ◽

Economic Losses ◽

Clinical Samples ◽

Diagnosis Method ◽

Pcr Method ◽

And Control

Abstract Background: African swine fever (ASF) is the most important disease to the pigs and cause serious economic losses to the countries with large-scale swine production. Vaccines are recognized as the most useful tool to prevent and control ASF virus (ASFV) infection. Currently, the MGF505 and MGF360 gene-deleted ASFVs or combined with CD2v deletion were confirmed to be the most promising vaccine candidates. Thus, it is essential to develop a diagnosis method to discriminate wide-type strain from the vaccines used.Results: In this study, we established a duplex TaqMan real-time PCR based on the B646L gene and MGF505-2R gene. The sequence alignment showed that the targeted regions of primers and probes are highly conserved in the genotype II ASFVs. The duplex real-time assay can specifically detect B646L and MGF505-2R gene single or simultaneously without cross-reaction with other porcine viruses tested. The limit of detection was 5.8 copies and 3.0 copies for the standard plasmids containing B646L and MGF505-2R genes, respectively. Clinical samples were tested in parallel by duplex real-time PCR and a commercial ASFV detection kit. The detection results of these two assays against B646L gene were well consistent.Conclusion: We successfully developed and evaluated a duplex TaqMan real-time PCR method which can effectively distinguish the wide type and MGF505 gene-deleted ASFVs. It would be a useful tool for the clinical diagnosis and control of ASF.

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Novel multiplex real-time PCR assays reveal a high prevalence of diarrhoeagenic Escherichia coli pathotypes in healthy and diarrhoeal children in the South of Vietnam

10.21203/rs.3.rs-18430/v1 ◽

2020 ◽

Author(s):

Vu Thuy Duong ◽

Le Thi Phuong Tu ◽

Ha Thanh Tuyen ◽

Le Thi Quynh Nhi ◽

James I Campbell ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Real Time Pcr ◽

Clinical Samples ◽

Middle Income ◽

Middle Income Countries ◽

Virulence Markers ◽

Low Middle Income Countries ◽

High Prevalence ◽

Pcr Assays

Abstract BackgroundDiarrhoeagenic Escherichia coli (DEC) infections are common in children in low-middle income countries (LMICs). However, detecting the various DEC pathotypes is complex as they cannot be differentiated by classical microbiology. We developed four multiplex real-time PCR assays were to detect virulence markers of six DEC pathotypes; specificity was tested using DEC controls and other enteric pathogens. PCR amplicons from the six E. coli pathotypes were purified and amplified to be used to optimize PCR reactions and to calculate reproducibility. After validation, these assays were applied to clinical samples from healthy and diarrhoeal Vietnamese children and associated with clinical data. ResultsThe multiplex real-time PCRs were found to be reproducible, and specific. At least one DEC variant was detected in 34.7% (978/2,815) of the faecal samples from diarrhoeal children; EAEC, EIEC and atypical EPEC were most frequent Notably, 41.2% (205/498) of samples from non-diarrhoeal children was positive with a DEC pathotype. In this population, only EIEC, which was detected in 34.3% (99/289) of diarrhoeal samples vs. 0.8% (4/498) non-diarrhoeal samples (p<0.001), was significantly associated with diarrhoea. Multiplex real-time PCR when applied to clinical samples is an efficient and high-throughput approach to DEC pathotypes. ConclusionsThis approach revealed high carriage rates of DEC pathotypes among Vietnamese children. We describe a novel diagnostic approach for DEC, which provides baseline data for future surveillance studies assessing DEC burden in LMICs.

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Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

Archives of Pathology & Laboratory Medicine ◽

10.5858/134.3.444 ◽

2010 ◽

Vol 134 (3) ◽

pp. 444-448 ◽

Cited By ~ 1

Author(s):

Zhengming Gu ◽

Jianmin Pan ◽

Matthew J. Bankowski ◽

Randall T. Hayden

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

Quantitative Polymerase Chain Reaction ◽

Bk Virus ◽

Clinical Samples ◽

Quantitative Detection ◽

Chain Reaction ◽

Pcr Method ◽

Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

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Introducing and validation of SYBR Green Real-Time PCR method to determinate sex ratio in bovine semen

Animal Reproduction Science ◽

10.1016/j.anireprosci.2013.05.009 ◽

2013 ◽

Vol 140 (1-2) ◽

pp. 1-6 ◽

Cited By ~ 3

Author(s):

Adham Fani Maleki ◽

AliReza Heravi Moussavi ◽

Mohammad Reza Nassiri ◽

Mojtaba Tahmoorespur ◽

Seyed Alireza Vakili

Keyword(s):

Sex Ratio ◽

Real Time ◽

Real Time Pcr ◽

Sybr Green ◽

Bovine Semen ◽

Pcr Method

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SYBR Green Real-Time PCR Method To Detect Clostridium botulinum Type A

Applied and Environmental Microbiology ◽

10.1128/aem.02234-06 ◽

2007 ◽

Vol 73 (9) ◽

pp. 2891-2896 ◽

Cited By ~ 31

Author(s):

Lucia Fenicia ◽

Fabrizio Anniballi ◽

Dario De Medici ◽

Elisabetta Delibato ◽

Paolo Aureli

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Clostridium Botulinum ◽

False Negative ◽

Type A ◽

Sybr Green ◽

Mouse Bioassay ◽

Chelex 100 ◽

Microbiological Methods ◽

Pcr Method

ABSTRACT Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 × 101 copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%).

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Real-Time PCR Assay for Detection and Differentiation of Shiga Toxin-Producing Escherichia coli from Clinical Samples

Journal of Clinical Microbiology ◽

10.1128/jcm.00115-15 ◽

2015 ◽

Vol 53 (7) ◽

pp. 2148-2153 ◽

Cited By ~ 28

Author(s):

Xuan Qin ◽

Eileen J. Klein ◽

Emmanouil Galanakis ◽

Anita A. Thomas ◽

Jennifer R. Stapp ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Primary Cultures ◽

Clinical Samples ◽

Pcr Assay ◽

Content Type ◽

E Coli ◽

Successful Recovery

Timely accurate diagnosis of Shiga toxin-producingEscherichia coli(STEC) infections is important. We evaluated a laboratory-developed real-time PCR (LD-PCR) assay targetingstx1,stx2, andrfbEO157with 2,386 qualifying stool samples submitted to the microbiology laboratory of a tertiary care pediatric center between July 2011 and December 2013. Broth cultures of PCR-positive samples were tested for Shiga toxins by enzyme immunoassay (EIA) (ImmunoCard STAT! enterohemorrhagicE. coli[EHEC]; Meridian Bioscience) and cultured in attempts to recover both O157 and non-O157 STEC.E. coliO157 and non-O157 STEC were detected in 35 and 18 cases, respectively. Hemolytic uremic syndrome (HUS) occurred in 12 patients (10 infected with STEC O157, one infected with STEC O125ac, and one with PCR evidence of STEC but no resulting isolate). Among the 59 PCR-positive STEC specimens from 53 patients, only 29 (54.7%) of the associated specimens were toxin positive by EIA. LD-PCR differentiated STEC O157 from non-O157 usingrfbEO157, and LD-PCR results prompted successful recovery ofE. coliO157 (n= 25) and non-O157 STEC (n= 8) isolates, although the primary cultures and toxin assays were frequently negative. A rapid “mega”-multiplex PCR (FilmArray gastrointestinal panel; BioFire Diagnostics) was used retrospectively, and results correlated with LD-PCR findings in 25 (89%) of the 28 sorbitol-MacConkey agar culture-negative STEC cases. These findings demonstrate that PCR is more sensitive than EIA and/or culture and distinguishes between O157 and non-O157 STEC in clinical samples and thatE. coliO157:H7 remains the predominant cause of HUS in our institution. PCR is highly recommended for rapid diagnosis of pediatric STEC infections.

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Absolute Quantitation of Different Genotypes of Hepatitis C Virus RNA in Clinical Samples by a Modified Real-Time PCR Method

Laboratory Medicine ◽

10.1309/lmhxo54sa8gypirh ◽

2011 ◽

Vol 42 (6) ◽

pp. 333-337

Author(s):

Karina del Carmen Trujillo-Murillo ◽

Diana Cristina Pérez-Ibave ◽

Clara Patricia Ríos-Ibarra ◽

Eda Guadalupe Ramirez-Valles ◽

Ana Rosa Rincón-Sánchez ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Real Time ◽

Real Time Pcr ◽

Clinical Samples ◽

Hepatitis C Virus Rna ◽

Absolute Quantitation ◽

Virus Rna ◽

Pcr Method

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Development and evaluation of duplex TaqMan real-time PCR assay for detection and differentiation of wide-type and MGF505-2R gene-deleted African swine fever viruses

BMC Veterinary Research ◽

10.1186/s12917-020-02639-2 ◽

2020 ◽

Vol 16 (1) ◽

Author(s):

Zhenhua Guo ◽

Kunpeng Li ◽

Songlin Qiao ◽

Xin-xin Chen ◽

Ruiguang Deng ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Large Scale ◽

Limit Of Detection ◽

African Swine Fever ◽

Economic Losses ◽

Clinical Samples ◽

Diagnosis Method ◽

Pcr Method ◽

And Control

Abstract Background African swine fever (ASF) is the most important disease to the pigs and cause serious economic losses to the countries with large-scale swine production. Vaccines are recognized as the most useful tool to prevent and control ASF virus (ASFV) infection. Currently, the MGF505 and MGF360 gene-deleted ASFVs or combined with CD2v deletion were confirmed to be the most promising vaccine candidates. Thus, it is essential to develop a diagnosis method to discriminate wide-type strain from the vaccines used. Results In this study, we established a duplex TaqMan real-time PCR based on the B646L gene and MGF505-2R gene. The sequence alignment showed that the targeted regions of primers and probes are highly conserved in the genotype II ASFVs. The duplex real-time assay can specifically detect B646L and MGF505-2R gene single or simultaneously without cross-reaction with other porcine viruses tested. The limit of detection was 5.8 copies and 3.0 copies for the standard plasmids containing B646L and MGF505-2R genes, respectively. Clinical samples were tested in parallel by duplex real-time PCR and a commercial ASFV detection kit. The detection results of these two assays against B646L gene were well consistent. Conclusion We successfully developed and evaluated a duplex TaqMan real-time PCR method which can effectively distinguish the wide type and MGF505 gene-deleted ASFVs. It would be a useful tool for the clinical diagnosis and control of ASF.

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Development and Evaluation of Duplex TaqMan Real-Time PCR Assay for Detection and Differentiation of Wide-Type and MGF505 Gene-Deleted African Swine Fever Viruses

10.21203/rs.3.rs-59710/v1 ◽

2020 ◽

Author(s):

Zhenhua Guo ◽

Kunpeng Li ◽

Songlin Qiao ◽

Xinxin Chen ◽

Ruiguang Deng ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Large Scale ◽

Limit Of Detection ◽

African Swine Fever ◽

Economic Losses ◽

Clinical Samples ◽

Diagnosis Method ◽

Pcr Method ◽

And Control

Abstract Background: African swine fever (ASF) is the most important disease to the pigs and cause serious economic losses to the countries with large-scale swine production. Vaccines are recognized as the most useful tool to prevent and control ASF virus (ASFV) infection. Currently, the MGF505 and MGF360 gene-deleted ASFVs or combined with CD2v deletion were confirmed to be the most promising vaccine candidates. Thus, it is essential to develop a diagnosis method to discriminate wide-type strain from the vaccines used. Results: In this study, we established a duplex TaqMan real-time PCR based on the B646L gene and MGF505-2R gene. The sequence alignment showed that the targeted regions of primers and probes are highly conserved in the genotype II ASFVs. The duplex real-time assay can specifically detect B646L and MGF505-2R gene single or simultaneously without cross-reaction with other porcine viruses tested. The limit of detection was 5.8 copies and 3.0 copies for the standard plasmids containing B646L and MGF505-2R genes, respectively. Clinical samples were tested in parallel by duplex real-time PCR and a commercial ASFV detection kit. The detection results of these two assays against B646L gene were well consistent. Conclusion: We successfully developed and evaluated a duplex TaqMan real-time PCR method which can effectively distinguish the wide type and MGF505 gene-deleted ASFVs. It would be a useful tool for the clinical diagnosis and control of ASF.

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