Quantitative detection method of Enterocytozoon hepatopenaei using TaqMan probe real-time PCR

Liu, Yamei; Qiu, Liu; Sheng, Anzhi; Wan, Xinyi; Cheng, Dongyuan; Huang, Jie

doi:10.1016/j.jip.2017.12.006

Cited by 67 publications

(37 citation statements)

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“…There are 2 approaches that use qPCR: 1 based on fluorescent dyes and 1 that is targeted at specific fluorescent‐labeled DNA sequences, called probes . The invention of TaqMan probes has overcome the limitations of SYBR Green I, that is, its lack of specificity owing to its ability to bind to all DNA double‐stranded structures . During the extension stage of TaqMan‐PCR, the DNA polymerase removes the TaqMan probe by hydrolysis, resulting in a fluorescent signal.…”

Section: Discussionmentioning

confidence: 99%

“…[60][61][62] The invention of TaqMan probes has overcome the limitations of SYBR Green I, that is, its lack of specificity owing to its ability to bind to all DNA double-stranded structures. [63][64][65][66] During the extension stage of TaqMan-PCR, the DNA polymerase removes the TaqMan probe by hydrolysis, resulting in a fluorescent signal. Moreover, multiple fluorescence signals can be used to detect the expression of several genes simultaneously using multiple channels.…”

Section: Discussionmentioning

confidence: 99%

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circMAN1A2 could serve as a novel serum biomarker for malignant tumors

et al. 2019

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Novel diagnostic and prognostic biomarkers of cancers are needed to improve precision medicine. Circular RNAs act as important regulators in cancers at the transcriptional and posttranscriptional levels. The circular RNA circMAN1A2 is highly expressed in nasopharyngeal carcinoma according to our previous RNA sequencing data; however, the expression and functions of circMAN1A2 in cancers are still obscure. Therefore, in this study, we evaluated the expression of circMAN1A2 in the sera of patients with nasopharyngeal carcinoma and other malignant tumors and analyzed its correlations with clinical features and diagnostic values. The expression levels of circMAN1A2 were detected by quantitative real‐time PCR, and the correlations of clinical features with circMAN1A2 expression were analyzed by χ2 tests. Receiver operating characteristic curves were used to evaluate the clinical applications of circMAN1A2. The results showed that circMAN1A2 was upregulated in nasopharyngeal carcinoma, oral cancer, thyroid cancer, ovarian cancer, and lung cancer, with areas under the curves of 0.911, 0.779, 0.734, 0.694, and 0.645, respectively, indicating the good diagnostic value of circMAN1A2. Overall, our findings suggested that circMAN1A2 could be a serum biomarker for malignant tumors, providing important insights into diagnostic approaches for malignant tumors. Further studies are needed to elucidate the mechanisms of circMAN1A2 in the pathogenesis of cancer.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

circMAN1A2 could serve as a novel serum biomarker for malignant tumors

et al. 2019

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“…Enterocytozoon hepatopenaei (EHP) is the microsporidian parasite that causes hepatopancreatic microsporidiosis in shrimp ( Chayaburakul et al, 2004 ; Tangprasittipap et al, 2013 ). Moreover, EHP is an emerging pathogen that affects the cultured shrimp Penaeus vannamei in many countries, such as Vietnam, Thailand, Brunei, Malaysia, Indonesia, China, and Venezuela ( Chayaburakul et al, 2004 ; Liu et al, 2017 ; Rajendran et al, 2016 ; Tang et al, 2015 , 2016 ; Tangprasittipap et al, 2013 ; Thi Ha et al, 2010 ), and is imposing a continuous threat to shrimp farming industries in future. The shrimp with EHP could be an immune risk factor, causing an increased susceptibility to pathogens of both acute hepatopancreatic necrosis disease and septic hepatopancreatic necrosis ( Aranguren, Han & Tang, 2017 ).…”

Section: Introductionmentioning

confidence: 99%

Real-time loop-mediated isothermal amplification for rapid detection of Enterocytozoon hepatopenaei

Cai

FanDe²,

Xu³

et al. 2018

PeerJ

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BackgroundEnterocytozoon hepatopenaei (EHP) is a newly emerged microsporidian parasite that causes retarded shrimp growth in many countries. But there are no effective approaches to control this disease to date. The EHP could be an immune risk factor for increased dissemination of other diseases. Further, EHP infection involves the absence of obvious clinical signs and it is difficult to identify the pathogen through visual examination, increasing the risk of disease dissemination. It is urgent and necessary to develop a specific, rapid and sensitive EHP-infected shrimp diagnostic method to detect this parasite. In the present study, we developed and evaluated a rapid real-time loop-mediated isothermal amplification (real-time LAMP) for detection of EHP.MethodsA rapid and efficient real-time LAMP method for the detection of EHP has been developed. Newly emerged EHP pathogens in China were collected and used as the sample, and three sets of specificity and sensitivity primers were designed. Three other aquatic pathogens were used as templates to test the specificity of the real-time LAMP assay. Also, we compared the real-time LAMP with the conventional LAMP by the serial dilutions of EHP DNA and their amplification curves. Application of real-time LAMP was carried out with clinical samples.ResultsPositive products were amplified only from EHP, but not from other tested species, EHP was detected from the clinical samples, suggesting a high specificity of this method. The final results of this assay were available within less than 45 min, and the initial amplification curve was observed at about 6 min. We found that the amplification with an exponential of sixfold dilutions of EHP DNA demonstrated a specific positive signal by the real-time LAMP, but not for the LAMP amplicons from the visual inspection. The real-time LAMP amplification curves demonstrated a higher slope than the conventional LAMP.DiscussionIn this study, pathogen virulence impacts have been increased in aquaculture and continuous observation was predominantly focused on EHP. The present study confirmed that the real-time LAMP assay is a promising and convenient method for the rapid identification of EHP in less time and cost. Its application greatly aids in the detection, surveillance, and prevention of EHP.

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“…PCR pro le was followed based on the published method [25]. For EHP quanti cation by real-time PCR, primers F:157 (5'-AGT AAA CTA TGC CGA CAA-3') and R:157 (5'-T TAA GCA GCA CAA TCC-3'), and a TaqMan probe (5-FAM-TCC TGG TAG TGT CCT TCC GT-TAMRA-3') were used following a previously published method [26].…”

Section: Histopathologymentioning

confidence: 99%

The Effect Of Salinity On Enterocytozoon Hepatopenaei  Infection In Penaeus Vannamei Under Experimental Conditions

Aranguren

Alghamdi

Belder

et al. 2020

Preprint

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Background: Enterocytozoon hepatopenaei (EHP) is an enteric pathogen that affects Penaeus vannamei and P. monodon shrimp in many SE Asian countries. In the western hemisphere, EHP was reported for the first time in 2016 in farmed P. vannamei in Venezuela. Anecdotal evidence suggests that EHP is more prevalent in grow-out ponds where the salinity is high (>15 parts per thousand (ppt) compared to grow-out ponds with low salinities (<5 ppt). Considering that P. vannamei is an euryhaline species, we were interested in knowing if EHP can propagate in P. vannamei in low salinities. Result: In this study, we described an experimental infection using fecal strings as a source inoculum. Specific Pathogen Free (SPF) P. vannamei were maintained at two different salinities including 2 ppt and 30 ppt, and continuously challenged using feces from known EHP-infected P. vannamei over a period of three weeks. The fecal strings, used as a source of EHP inocula in the challenges, was sufficient to elicit infection in shrimp maintained at both salinities. Infectivity of EHP in shrimp reared at 2 and 30 ppt salinities was confirmed by PCR and histopathology. The prevalence and the severity of EHP infection was higher at 30 ppt than 2 ppt. Conclusion: The data suggest that fecal strings is a reliable method to conduct EHP challenges via fecal-oral route. EHP infection can occur at a salinity as low as 2 ppt, although the prevalence and the severity of EHP infection is higher at a salinity of 30 ppt compared to 2 ppt.

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Quantitative detection method of Enterocytozoon hepatopenaei using TaqMan probe real-time PCR

Cited by 67 publications

References 5 publications

circMAN1A2 could serve as a novel serum biomarker for malignant tumors

circMAN1A2 could serve as a novel serum biomarker for malignant tumors

Real-time loop-mediated isothermal amplification for rapid detection of Enterocytozoon hepatopenaei

The Effect Of Salinity On Enterocytozoon Hepatopenaei  Infection In Penaeus Vannamei Under Experimental Conditions

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Quantitative detection method of Enterocytozoon hepatopenaei using TaqMan probe real-time PCR

Cited by 67 publications

References 5 publications

circMAN1A2 could serve as a novel serum biomarker for malignant tumors

circMAN1A2 could serve as a novel serum biomarker for malignant tumors

Real-time loop-mediated isothermal amplification for rapid detection of Enterocytozoon hepatopenaei

The Effect Of Salinity On Enterocytozoon Hepatopenaei &nbsp;Infection In Penaeus Vannamei Under Experimental Conditions

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The Effect Of Salinity On Enterocytozoon Hepatopenaei Infection In Penaeus Vannamei Under Experimental Conditions