Development of a novel real-time polymerase chain reaction assay for the sensitive detection of Schistosoma japonicum in human stool

Background Elimination and control of Schistosoma japonicum, the most virulent of the schistosomiasis-causing blood flukes, requires the development of sensitive and specific diagnostic tools capable of providing an accurate measurement of the infection prevalence in endemic areas. Typically, detection of S. japonicum has occurred using the Kato-Katz technique, but this methodology, which requires skilled microscopists, has been shown to radically underestimate levels of infection. With the ever-improving capabilities of next-generation sequencing and bioinformatic analysis tools, identification of satellite sequences and other highly repetitive genomic elements for use as real-time PCR diagnostic targets is becoming increasingly common. Assays developed using these targets have the ability to improve the sensitivity and specificity of results for epidemiological studies that can in turn be used to inform mass drug administration and programmatic decision making. Methodology/Principal findings Utilizing Tandem Repeat Analyzer (TAREAN) and RepeatExplorer2, a cluster-based analysis of the S. japonicum genome was performed and a tandemly arranged genomic repeat, which we named SjTR1 (Schistosoma japonicum Tandem Repeat 1), was selected as the target for a real-time PCR diagnostic assay. Based on these analyses, a primer/probe set was designed and the assay was optimized. The resulting real-time PCR test was shown to reliably detect as little as 200 ag of S. japonicum genomic DNA and as little as 1 egg per gram of human stool. Based on these results, the index assay reported in this manuscript is more sensitive than previously published real-time PCR assays for the detection of S. japonicum. Conclusions/Significance The extremely sensitive and specific diagnostic assay described in this manuscript will facilitate the accurate detection of S. japonicum, particularly in regions with low levels of endemicity. This assay will be useful in providing data to inform programmatic decision makers, aiding disease control and elimination efforts.

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Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽

10.3390/pathogens10020188 ◽

2021 ◽

Vol 10 (2) ◽

pp. 188

Author(s):

Tanja Hoffmann ◽

Andreas Hahn ◽

Jaco J. Verweij ◽

Gérard Leboulle ◽

Olfert Landt ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Pcr Assay ◽

Bead Beating ◽

Stool Samples ◽

Pcr Assays ◽

Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

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The circRAB3IP Mediated by eIF4A3 and LEF1 Contributes to Enzalutamide Resistance in Prostate Cancer by Targeting miR-133a-3p/miR-133b/SGK1 Pathway

Frontiers in Oncology ◽

10.3389/fonc.2021.752573 ◽

2021 ◽

Vol 11 ◽

Author(s):

Dong Chen ◽

Yaqin Wang ◽

Feiya Yang ◽

Adili Keranmu ◽

Qingxin Zhao ◽

...

Keyword(s):

Prostate Cancer ◽

Real Time ◽

Expression Pattern ◽

Real Time Pcr ◽

Bioinformatic Analysis ◽

Biological Functions ◽

Quantitative Real Time Pcr ◽

Regulatory Effects

An increasing number of studies have shown that circRNAs are closely related to the carcinogenesis and development of prostate cancer (PCa). However, little is known about the effect of the biological functions of circRNAs on the enzalutamide resistance of PCa. Through bioinformatic analysis and experiments, we investigated the expression pattern of circRNAs in enzalutamide-resistant PCa cells. Quantitative real-time PCR was used to detect the expression of circRAB3IP, and plasmids that knock down or overexpress circRAB3IP were used to evaluate its effect on the enzalutamide sensitivity of PCa cells. Mechanistically, we explored the potential regulatory effects of eIF4A3 and LEF1 on the biogenesis of circRAB3IP. Our in vivo and in vitro data indicated that increased expression of circRAB3IP was found in enzalutamide-resistant PCa, and knockdown of circRAB3IP significantly enhanced enzalutamide sensitivity in PCa cells. However, upregulation of circRAB3IP resulted in the opposite effects. Further mechanistic research demonstrated that circRAB3IP could regulate the expression of serum and glucocorticoid-regulated kinase 1 (SGK1) by serving as a sponge that directly targets miR-133a-3p/miR-133b. Then, we showed that circRAB3IP partially exerted its biological functions via SGK1 signaling. Furthermore, we discovered that eIF4A3 and LEF1 might increase circRAB3IP expression in PCa.

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Development of Conventional Multiplex PCR: A Rapid Technique for Simultaneous Detection of Soil-Transmitted Helminths

Pathogens ◽

10.3390/pathogens8030152 ◽

2019 ◽

Vol 8 (3) ◽

pp. 152 ◽

Cited By ~ 2

Author(s):

Vivornpun Sanprasert ◽

Ruthairat Kerdkaew ◽

Siriporn Srirungruang ◽

Sarit Charuchaibovorn ◽

Kobpat Phadungsaksawasdi ◽

...

Keyword(s):

Multiplex Pcr ◽

Real Time ◽

Real Time Pcr ◽

Intestinal Parasites ◽

Simultaneous Detection ◽

Diagnostic Tools ◽

Multiple Infections ◽

Parasitic Infections ◽

Soil Transmitted Helminths ◽

Stool Samples

Soil-transmitted helminths (STHs) are the most common intestinal parasites infecting humans worldwide. STH infections are a major cause of morbidity and disability. Accurate diagnostic tools are pivotal for assessing the exact prevalence of parasitic infections. Microscopic examination and culture techniques have been used to observe the presence of eggs or larvae of parasites in stool samples, but they are time-consuming and have low sensitivity. Therefore, accurate, simple, and inexpensive diagnostic techniques are still required for simultaneous detection of STH infections. Although molecular-based techniques, such as real-time PCR and multiplex real-time PCR, have been developed, they are not suitable for routine diagnosis due to the requirement for expensive reagents and instruments. In this study, we established a conventional multiplex PCR for simultaneous rapid detection of Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis in stool samples. Our results show that the multiplex PCR could detect the DNA of STHs at a very low target gene concentrations (lower than 1 pg) with no cross-amplification. Multiplex PCR had five times higher sensitivity than the formalin–ethyl acetate concentration technique (FECT) in the detection of multiple infections, and two times higher for detection of S. stercoralis. However, multiplex PCR was comparable to FECT in the detection of A. lumbricoides and N. americanus. In conclusion, this method could be used as an alternative method for the detection of STHs, especially for S. stercoralis.

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Development of a sensitive and quantitative diagnostic assay for fish nervous necrosis virus based on two-target real-time PCR

Veterinary Microbiology ◽

10.1016/j.vetmic.2005.07.014 ◽

2005 ◽

Vol 110 (3-4) ◽

pp. 167-179 ◽

Cited By ~ 52

Author(s):

L DALLAVALLE ◽

V TOFFOLO ◽

M LAMPRECHT ◽

C MALTESE ◽

G BOVO ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Nervous Necrosis Virus ◽

Diagnostic Assay ◽

Necrosis Virus

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Diagnosis and epidemiological studies of human metapneumovirus using real-time PCR

Journal of Clinical Virology ◽

10.1016/j.jcv.2007.08.004 ◽

2007 ◽

Vol 40 (3) ◽

pp. 186-192 ◽

Cited By ~ 27

Author(s):

Kanti Pabbaraju ◽

Sallene Wong ◽

Thomas McMillan ◽

Bonita E. Lee ◽

Julie D. Fox

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Epidemiological Studies ◽

Human Metapneumovirus

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Novel Multiplex Real-Time PCR Diagnostic Assay for Identification and Differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis Complex Strains

Journal of Clinical Microbiology ◽

10.1128/jcm.01426-10 ◽

2010 ◽

Vol 49 (2) ◽

pp. 651-657 ◽

Cited By ~ 17

Author(s):

K. Reddington ◽

J. O'Grady ◽

S. Dorai-Raj ◽

M. Maher ◽

D. van Soolingen ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Real Time ◽

Real Time Pcr ◽

Mycobacterium Tuberculosis Complex ◽

Diagnostic Assay ◽

Tuberculosis Complex

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Preliminary validation of real-time PCR assays for the identification of Yersinia pestis

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm.2008.251 ◽

2008 ◽

Vol 46 (9) ◽

Cited By ~ 14

Author(s):

Herbert Tomaso ◽

Daniela Jacob ◽

Meike Eickhoff ◽

Holger C. Scholz ◽

Sascha Al Dahouk ◽

...

Keyword(s):

Yersinia Pestis ◽

Real Time ◽

Real Time Pcr ◽

Rapid Identification ◽

Diagnostic Tools ◽

The Real ◽

Preliminary Validation ◽

Pcr Assays

Abstract:: A total of four samples of quantified: All participating laboratories were able to perform the real-time PCR assays according to the protocols provided and identified the samples containing: Our real-time PCR system proved to be highly reproducible and has the potential of complementing the diagnostic tools for rapid identification ofClin Chem Lab Med 2008;46:1239–44.

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Use of Multiplex Real-Time PCR To Diagnose Scrub Typhus

Journal of Clinical Microbiology ◽

10.1128/jcm.02181-16 ◽

2017 ◽

Vol 55 (5) ◽

pp. 1377-1387 ◽

Cited By ~ 12

Author(s):

Wiwit Tantibhedhyangkul ◽

Ekkarat Wongsawat ◽

Saowaluk Silpasakorn ◽

Duangdao Waywa ◽

Nuttawut Saenyasiri ◽

...

Keyword(s):

Early Diagnosis ◽

Real Time ◽

Real Time Pcr ◽

Scrub Typhus ◽

Febrile Illness ◽

Asia Pacific ◽

Diagnostic Tools ◽

Pcr Assay ◽

Content Type ◽

Serologic Tests

ABSTRACTScrub typhus, caused byOrientia tsutsugamushi, is a common cause of acute undifferentiated febrile illness in the Asia-Pacific region. However, its nonspecific clinical manifestation often prevents early diagnosis. We propose the use of PCR and serologic tests as diagnostic tools. Here, we developed a multiplex real-time PCR assay using hydrolysis (TaqMan) probes targetingO. tsutsugamushi47-kDa,groEL, and human interferon beta (IFN-β gene) genes to improve early diagnosis of scrub typhus. The amplification efficiency was higher than 94%, and the lower detection limit was 10 copies per reaction. We used a human gene as an internal DNA quality and quantity control. To determine the sensitivity of this PCR assay, we selected patients with confirmed scrub typhus who exhibited a clear 4-fold increase in the level of IgG and/or IgM. The PCR assay result was positive in 45 of 52 patients, indicating a sensitivity of 86.5% (95% confidence interval [CI]: 74.2 to 94.4). The PCR assessment was negative for all 136 non-scrub typhus patients, indicating a specificity of 100% (95% CI: 97.3 to 100). In addition, this test helped diagnose patients with inconclusive immunofluorescence assay (IFA) results and using single blood samples. In conclusion, the real-time PCR assay proposed here is sensitive and specific in diagnosing scrub typhus. Combining PCR and serologic tests will improve the diagnosis of scrub typhus among patients presenting with acute febrile illness.

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