Development of a Recombinase Polymerase Amplification Assay for Schistosomiasis Japonica Diagnosis in the Experimental Mice and Domestic Goats

Although the prevalence of schistosomiasis japonica has declined gradually in China, more accurate and sensitive diagnostic methods are urgently needed for the prevention and control of this disease. Molecular diagnostic methods are advantageous in terms of sensitivity and specificity, but they are time-consuming and require expensive instruments and skilled personnel, which limits their application in low-resource settings. In this study, an isothermal DNA amplification assay and recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD) were set up. It was used to detect S. japonicum infections in experimental mice and domestic goats by amplifying a specific DNA fragment of S. japonicum. The lower limit of detection for the LFD-RPA assay was evaluated using dilutions of plasmid containing the target sequence. Cross-reactivity was evaluated using genomic DNA from eight other parasites. The effectiveness of the LFD-RPA assay was verified by assessing 36 positive plasma samples and 36 negative plasma samples from mice. The LFD-RPA assay and real-time PCR were also used to assess 48 schistosomiasis japonica-positive plasma samples and 53 negative plasma samples from goats. The LFD-RPA assay could detect 2.6 femtogram (fg) of S. japonicum target DNA (~39 fg genomic DNA of S. japonicum), only 10-fold less sensitive than real-time PCR assay. There was no cross-reactivity with DNA from the other eight parasites, such as Haemonchus contortus and Spirometra. The whole amplification process could be completed within 15 min at 39°C, and the results can be observed easily using the LFD. The sensitivity and specificity of the LFD-RPA assay were 97.22% (35/36, 95% CI, 85.47%–99.93%) and 100% (36/36, 95% CI, 90.26%–100%) in mice, and 93.75% (45/48, 95% CI, 82.80%–98.69%) and 100% (53/53, 95% CI, 93.28%–100%) in goats. By comparison, the sensitivity and specificity of real-time PCR were 100% (36/36, 95% CI, 90.26%–100%) and 100% (36/36, 95% CI, 90.26%–100%) for mice, and 97.92% (47/48, 95% CI, 88.93%–99.95%) and 100% (53/53, 95% CI, 93.28%–100%) for goats. The LFD-RPA assay exhibits high sensitivity and specificity for the diagnosis of schistosomiasis japonica, and it is an alternative method for diagnosis schistosomiasis japonica in low resource setting.

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Challenges in laboratory diagnosis of Malaria in a low resource country among cases of acute febrile illness at tertiary care hospital in eastern Nepal: Comparative study on Conventional Vs Molecular approach

10.21203/rs.3.rs-35005/v1 ◽

2020 ◽

Author(s):

Pragyan Dahal ◽

Basudha Khanal ◽

Keshav Rai ◽

Vivek Kattel ◽

Satish Yadav ◽

...

Keyword(s):

Real Time ◽

Sensitivity And Specificity ◽

Malaria Elimination ◽

Real Time Pcr ◽

Gold Standard ◽

Febrile Illness ◽

Acute Febrile Illness ◽

Low Resource ◽

Elimination Programme ◽

Low Resource Country

Abstract Background For ongoing malaria elimination programme, available methods like microscopy and rapid diagnostic test (RDT) are not able to detect all the cases of malaria in acute febrile illness. These methods are entirely dependent on the course of infection, parasite load and skilled technical resources thus the study was carried out to compare performance of microscopy and RDT which are commonly used in a low resource country along with reference to real-time PCR. Methods Altogether 52 blood samples collected from patients with acute febrile illness were tested by microscopy, RDT and real-time PCR. The results were compared in terms of sensitivity and specificity. Results The test results were as follows: 5.8% positivity by Microscopy, 13.5% positivity by RDT and 27% by real time PCR. Taking into consideration of PCR as a gold standard method microscopy showed 21.4% sensitivity and 100% specificity. Likewise, RDT results revealed 28.6% sensitivity and 92.1% specificity. Conclusion Despite of various diagnostic tools available, microscopy stills remains the gold standard for the diagnosis and RDT is the user friendly to execute the test under the tree, but our preliminary results emphasized the need to implement the test with the higher sensitivity and specificity in context of malaria elimination programme which could be another important opportunity to understand the parasite circulation in case of low endemic region. However, these results should be further verified with the large study cohort in order to document the submicroscopic infection.

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A Novel Real-Time PCR Assay for the Rapid Detection of Virulent Streptococcus equi Subspecies zooepidemicus—An Emerging Pathogen of Swine

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.604675 ◽

2021 ◽

Vol 8 ◽

Author(s):

Suresh V. Kuchipudi ◽

Meera Surendran Nair ◽

Michele Yon ◽

Abhinay Gontu ◽

Ruth H. Nissly ◽

...

Keyword(s):

Real Time ◽

Sensitivity And Specificity ◽

High Mortality ◽

Real Time Pcr ◽

Diagnostic Methods ◽

Clinical Samples ◽

Pcr Assay ◽

Streptococcus Equi ◽

Swine Herds ◽

Pcr Targeting

Streptococcus equi subspecies zooepidemicus, a zoonotic bacterial pathogen caused a series of outbreaks with high mortality affecting swine herds in multiple locations of the USA and Canada in 2019. Further genetic analysis revealed that this agent clustered with ATCC 35246, a S. zooepidemicus strain associated with high mortality outbreaks in swine herds of China originally reported in 1977. Rapid and accurate diagnosis is absolutely critical for controlling and limiting further spread of this emerging disease of swine. Currently available diagnostic methods including bacteriological examination and PCR assays do not distinguish between the virulent strains and avirulent commensal strains of S. zooepidemicus, which is critical given that this pathogen is a normal inhabitant of the swine respiratory tract. Based on comparative analyses of whole genome sequences of the virulent isolates and avirulent sequences, we identified a region in the SzM gene that is highly conserved and restricted to virulent S. zooepidemicus strains. We developed and validated a novel probe-based real-time PCR targeting the conserved region of SzM. The assay was highly sensitive and specific to the virulent swine isolates of Streptococcus equi subspecies zooepidemicus. No cross reactivity was observed with avirulent S. zooepidemicus isolates as well as other streptococcal species and a panel of porcine respiratory bacterial and viral pathogens. The PCR efficiency of the assay was 96.64 % and was able to detect as little as 20 fg of the bacterial DNA. We then validated the diagnostic sensitivity and specificity of the new PCR assay using a panel of clinical samples (n = 57) and found that the assay has 100% sensitivity and specificity as compared to bacteriological culture method. In summary, the PCR assay will be an extremely valuable tool for the rapid accurate detection of virulent swine S. zooepidemicus isolates and directly from clinical samples.

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Evaluation of a real-time PCR assay for diagnosis of schistosomiasis japonica in the domestic goat

Parasites & Vectors ◽

10.1186/s13071-020-04420-8 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Qinghong Guo ◽

Cheng Chen ◽

Keke Zhou ◽

Yugang Li ◽

Laibao Tong ◽

...

Keyword(s):

Real Time ◽

Sensitivity And Specificity ◽

Real Time Pcr ◽

Disease Transmission ◽

Schistosomiasis Japonica ◽

Enzyme Linked Immunosorbent Assay ◽

Pcr Assay ◽

Serum Samples ◽

Endemic Region ◽

Pcr Method

Abstract Background Schistosomiasis japonica is an infectious disease caused by Schistosoma japonicum that seriously endangers human health. Domestic animals have important roles in disease transmission and goats are considered a primary reservoir host and source of infection. The prevalence and intensity of schistosomiasis infections have significantly decreased in China, and a more sensitive, specific detection method is urgently needed. The aim of this study was to develop a real-time PCR assay for accurate detection of S. japonicum infection in goats. Methods A real-time PCR method for detecting schistosomiasis japonica in goats was developed by amplification of a specific S. japonicum DNA fragment, and validated using a total of 94 negative and 159 positive plasma and serum samples collected in our previous study of S. japonicum infection. Both plasma and serum samples were evaluated by real-time PCR and enzyme-linked immunosorbent assay (ELISA). In addition, 120 goat plasma samples from an S. japonicum-endemic area (Wangjiang) and 33 from a non-endemic region (Weihai) were collected and evaluated using our method. Results The sensitivity and specificity of the real-time PCR for detecting infected samples were 98.74% (157/159, 95% CI: 95.53–99.85%) and 100% (94/94, 95% CI: 96.15–100%), respectively. For the ELISA, sensitivity and specificity were 98.11% (156/159, 95% CI: 94.59–99.61%) and 90.43% (85/94, 95% CI: 82.60–95.53%), respectively. Further, we found positivity rates for S. japonicum infection in Wangjiang and Weihai of 8.33% (10/120, 95% CI: 4.07–14.79%) and 0% (0/33, 95% CI: 0–10.58%), respectively. Conclusions The results of this study indicate that our real-time PCR method exhibits higher sensitivity and specificity than ELISA and is a useful method for detection of S. japonicum infection in goats.

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Multiplex Real-Time PCR Assay for Simultaneous Identification of Neisseria gonorrhoeae and Its Ciprofloxacin Susceptibility Status

Journal of Clinical Microbiology ◽

10.1128/jcm.00855-17 ◽

2017 ◽

Vol 55 (11) ◽

pp. 3201-3209 ◽

Cited By ~ 11

Author(s):

Sumudu R. Perera ◽

Nurul H. Khan ◽

Irene Martin ◽

Ali Taheri ◽

Rajinder P. Parti ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Real Time ◽

Sensitivity And Specificity ◽

Real Time Pcr ◽

Genomic Dna ◽

Rt Pcr ◽

Pcr Assay ◽

Content Type ◽

Simultaneous Identification ◽

Susceptibility Status

ABSTRACTA real-time PCR (RT-PCR) assay was designed for the simultaneous identification ofNeisseria gonorrhoeaeand its ciprofloxacin susceptibility status. A SYBR green-based multiplex RT-PCR format was used; it comprised two different forward primers and a common reverse primer to detect single nucleotide polymorphisms (SNPs) ingyrAofN. gonorrhoeae. The primer pairs were evaluated for their sensitivity and specificity using genomic DNA from 254N. gonorrhoeaeisolates (82 were ciprofloxacin susceptible and 172 were ciprofloxacin resistant) and 23 non-N. gonorrhoeaespecies isolates. The performance of the primers was validated using genomic DNA from 100 differentN. gonorrhoeaeisolates (46 were ciprofloxacin susceptible and 54 were ciprofloxacin resistant) and 52 non-N. gonorrhoeaeisolates. The latter panel was revalidated by testing 99 (46 isolates were ciprofloxacin susceptible and 53 isolates were ciprofloxacin resistant) of theN. gonorrhoeaeisolates and 23 non-N. gonorrhoeaeisolates. These primers detectedN. gonorrhoeaeand its ciprofloxacin susceptibility status with over 99% sensitivity and specificity for all panels tested. This assay has the potential to be an inexpensive and rapid test for the simultaneous identification ofN. gonorrhoeaeand its ciprofloxacin susceptibility status.

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Detection of Biological Threat Agents by Real-Time PCR: Comparison of Assay Performance on the R.A.P.I.D., the LightCycler, and the Smart Cycler Platforms

Clinical Chemistry ◽

10.1373/clinchem.2005.052522 ◽

2006 ◽

Vol 52 (1) ◽

pp. 141-145 ◽

Cited By ~ 69

Author(s):

Deanna R Christensen ◽

Laurie J Hartman ◽

Bonnie M Loveless ◽

Melissa S Frye ◽

Michelle A Shipley ◽

...

Keyword(s):

Real Time ◽

Sensitivity And Specificity ◽

Real Time Pcr ◽

Limit Of Detection ◽

Dna Amplification ◽

Pcr Primers ◽

Cross Reactivity ◽

Dna Templates ◽

Assay Performance ◽

Fluorogenic Probes

Abstract Background: Rapid detection of biological threat agents is critical for timely therapeutic administration. Fluorogenic PCR provides a rapid, sensitive, and specific tool for molecular identification of these agents. We compared the performance of assays for 7 biological threat agents on the Idaho Technology, Inc. R.A.P.I.D.®, the Roche LightCycler®, and the Cepheid Smart Cycler®. Methods: Real-time PCR primers and dual-labeled fluorogenic probes were designed to detect Bacillus anthracis, Brucella species, Clostridium botulinum, Coxiella burnetii, Francisella tularensis, Staphylococcus aureus, and Yersinia pestis. DNA amplification assays were optimized by use of Idaho Technology buffers and deoxynucleotide triphosphates supplemented with Invitrogen Platinum® Taq DNA polymerase, and were subsequently tested for sensitivity and specificity on the R.A.P.I.D., the LightCycler, and the Smart Cycler. Results: Limit of detection experiments indicated that assay performance was comparable among the platforms tested. Exclusivity and inclusivity testing with a general bacterial nucleic acid cross-reactivity panel containing 60 DNAs and agent-specific panels containing nearest neighbors for the organisms of interest indicated that all assays were specific for their intended targets. Conclusion: With minor supplementation, such as the addition of Smart Cycler Additive Reagent to the Idaho Technology buffers, assays for DNA templates from biological threat agents demonstrated similar performance, sensitivity, and specificity on all 3 platforms.

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Improving molecular detection of Candida DNA in whole blood: comparison of seven fungal DNA extraction protocols using real-time PCR

Journal of Medical Microbiology ◽

10.1099/jmm.0.47617-0 ◽

2008 ◽

Vol 57 (3) ◽

pp. 296-303 ◽

Cited By ~ 29

Author(s):

L. Metwally ◽

D. J. Fairley ◽

P. V. Coyle ◽

R. J. Hay ◽

S. Hedderwick ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Dna Extraction ◽

Real Time Pcr ◽

Whole Blood ◽

Genomic Dna ◽

Molecular Techniques ◽

Diagnostic Methods ◽

Yeast Cells ◽

Pcr Assay

The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood.

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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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Evaluation of the New BD Max GC Real-Time PCR Assay, Analytically and Clinically as a Supplementary Test for the BD ProbeTec GC Qx Amplified DNA Assay, for Molecular Detection of Neisseria gonorrhoeae

Journal of Clinical Microbiology ◽

10.1128/jcm.01962-15 ◽

2015 ◽

Vol 53 (12) ◽

pp. 3935-3937 ◽

Cited By ~ 5

Author(s):

Daniel Golparian ◽

Stina Boräng ◽

Martin Sundqvist ◽

Magnus Unemo

Keyword(s):

Neisseria Gonorrhoeae ◽

Real Time ◽

Sensitivity And Specificity ◽

Real Time Pcr ◽

Molecular Detection ◽

Analytical Sensitivity ◽

Pcr Assay ◽

Content Type ◽

Dna Assay ◽

Supplementary Test

The new BD Max GC real-time PCR assay showed high clinical and analytical sensitivity and specificity. It can be an effective and accurate supplementary test for the BD ProbeTec GC Qx amplified DNA assay, which had suboptimal specificity, and might also be used for initial detection ofNeisseria gonorrhoeae.

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