Objective:To develop a rapid,cost effective RT-PCR method for the mass scale diagnosis of such diseases at the vireraia stage to find out the actual disease burden in that area.Methods:For this purpose,cases with the ...Objective:To develop a rapid,cost effective RT-PCR method for the mass scale diagnosis of such diseases at the vireraia stage to find out the actual disease burden in that area.Methods:For this purpose,cases with the history of only short febrile illness were considered.Thus 157 samples with the history of dengue/chikungunya like illness and only 58 samples with a history of acute encephalitis syndrome(AES)were selected.Results:Out of 157 samples,42 and 74 were detected as dengue and chikungunya,respectively and out of 58 AES cases only 23 could be detected as Japanese encephalitis by this RT-PCR method.Conclusions:This cost effective RT-PCR method can detect the total positive cases that remain undetected by EL1SA method.Moreover,this method is capable to detect the viral RNA from patients'sera even after the appearance of IgM antibody at one fifth costs as compared with the other commercially available kits.展开更多
Dolphin Morbillivirus (DMV) is one of the most frequently detected pathogens in stranded cetacean specimens worldwide as well as in Italy. Due to the persistence of DMV in the Mediterranean Sea and to the lack of info...Dolphin Morbillivirus (DMV) is one of the most frequently detected pathogens in stranded cetacean specimens worldwide as well as in Italy. Due to the persistence of DMV in the Mediterranean Sea and to the lack of information about the efficiency of the available diagnostic techniques, the Italian National Reference Centre for diagnostic activities on dead stranded marine mammals (C.Re.Di.Ma) performed the first inter-laboratory ring trial with the aim to standardize a diagnostic biomolecular approach for DMV in Italy. Viral isolation is usually considered the “gold standard” for the definitive diagnosis of most pathogens, but it is not often feasible in DMV diagnosis, due to the poor preservation of virus-targeted tissues in stranded cetacean carcasses, as well as to the lack of appropriate sensitivity of cell lines towards DMV variability. Therefore direct viral detection on tissues by means of reverse transcription-PCR (RT-PCR) represents a valuable option for DMV infection’s diagnosis. For detecting DMV in cetacean die-offs occurred in the Mediterranean basin since 2013, C.Re.Di.Ma developed an RT-PCR based method targeting to a 287 bp fragment of DMV nucleoprotein (N) gene. With the purpose to evaluate its performances in terms of accuracy (Se = sensitivity and Sp = specificity) and precision (reproducibility), it was submitted to a ring trial. So, 12 Public Laboratories belonging to the Italian dead stranded marine mammals diagnostic network were asked to analyze a panel of 40 samples (positive and negative for DMV, using different dilutions of a viral suspension obtained from a cell culture supernatant of a DMV strain) with the aforementioned technique. Furthermore, we also aimed at comparing the accuracy of other 7 molecular methods routinely applied for DMV detection in Italy. For this purpose, the second panel of identical 40 DMV +ve and -ve samples was provided to Laboratories that routinely used DMV detection methods other than those developed by C.Re.Di.Ma, in order to be analyzed simultaneously with the method they usually applied. The C.Re.Di.Ma technique showed high accuracy [mean Se = 97.8% (95% CI 84.2% - 99.3%), mean Sp = 98.1% (95% CI 72.5% - 99.9%)] and very good precision [k combined equal to 0.91 (95% CI 0.87 - 0.95)]. In conclusion, this study highlighted a satisfactory reliability of most of the molecular methods used in Italy for DMV detection.展开更多
Noroviruses (NoVs) are widespread causes of nonbacterial gastroenteritis. Outbreaks of NoVs caused diseases are commonly ascribed to the consumption of contaminated shellfish. The concentration and RNA extraction of N...Noroviruses (NoVs) are widespread causes of nonbacterial gastroenteritis. Outbreaks of NoVs caused diseases are commonly ascribed to the consumption of contaminated shellfish. The concentration and RNA extraction of NoVs are crucial steps of detecting NoVs in shellfish. This study aimed to select a simple, rapid and highly efficient recovery method of NoVs detection with real-time RT-PCR. Four methods of recovering GI.3 and GII.4 NoVs from spiked digestive tissues of oysters and clams, respectively, were compared, of them, the method involving proteinase K and PEG 8000 was found the most efficient. With this method, 9.3% and 13.1% of GI.3 and GII.4 NoVs were recovered from oysters and 9.6% and 12.3% of GI.3 and GII.4 NoVs were recovered from clams, respectively. This method was further used to detect NoVs in 84 oysters (Crassostrea gigas) and 86 clams (Ruditapes philippinarum) collected from 10 coastal cities in China from Jan. 2011 to Feb. 2012. The NoVs isolation rates were 10.47% of clams (9/86) and 7.14% of oysters (6/84). All the detected NoVs belonged to genotype GII. The NoVs recovery method selected is efficient for NoVs detection in oysters and clams.展开更多
基金supported by the Department of Science and Technology,Goverment of West Bengal.India[grant No.705(Sanc.)ST/P/S&T/9G-27/2007]
文摘Objective:To develop a rapid,cost effective RT-PCR method for the mass scale diagnosis of such diseases at the vireraia stage to find out the actual disease burden in that area.Methods:For this purpose,cases with the history of only short febrile illness were considered.Thus 157 samples with the history of dengue/chikungunya like illness and only 58 samples with a history of acute encephalitis syndrome(AES)were selected.Results:Out of 157 samples,42 and 74 were detected as dengue and chikungunya,respectively and out of 58 AES cases only 23 could be detected as Japanese encephalitis by this RT-PCR method.Conclusions:This cost effective RT-PCR method can detect the total positive cases that remain undetected by EL1SA method.Moreover,this method is capable to detect the viral RNA from patients'sera even after the appearance of IgM antibody at one fifth costs as compared with the other commercially available kits.
文摘Dolphin Morbillivirus (DMV) is one of the most frequently detected pathogens in stranded cetacean specimens worldwide as well as in Italy. Due to the persistence of DMV in the Mediterranean Sea and to the lack of information about the efficiency of the available diagnostic techniques, the Italian National Reference Centre for diagnostic activities on dead stranded marine mammals (C.Re.Di.Ma) performed the first inter-laboratory ring trial with the aim to standardize a diagnostic biomolecular approach for DMV in Italy. Viral isolation is usually considered the “gold standard” for the definitive diagnosis of most pathogens, but it is not often feasible in DMV diagnosis, due to the poor preservation of virus-targeted tissues in stranded cetacean carcasses, as well as to the lack of appropriate sensitivity of cell lines towards DMV variability. Therefore direct viral detection on tissues by means of reverse transcription-PCR (RT-PCR) represents a valuable option for DMV infection’s diagnosis. For detecting DMV in cetacean die-offs occurred in the Mediterranean basin since 2013, C.Re.Di.Ma developed an RT-PCR based method targeting to a 287 bp fragment of DMV nucleoprotein (N) gene. With the purpose to evaluate its performances in terms of accuracy (Se = sensitivity and Sp = specificity) and precision (reproducibility), it was submitted to a ring trial. So, 12 Public Laboratories belonging to the Italian dead stranded marine mammals diagnostic network were asked to analyze a panel of 40 samples (positive and negative for DMV, using different dilutions of a viral suspension obtained from a cell culture supernatant of a DMV strain) with the aforementioned technique. Furthermore, we also aimed at comparing the accuracy of other 7 molecular methods routinely applied for DMV detection in Italy. For this purpose, the second panel of identical 40 DMV +ve and -ve samples was provided to Laboratories that routinely used DMV detection methods other than those developed by C.Re.Di.Ma, in order to be analyzed simultaneously with the method they usually applied. The C.Re.Di.Ma technique showed high accuracy [mean Se = 97.8% (95% CI 84.2% - 99.3%), mean Sp = 98.1% (95% CI 72.5% - 99.9%)] and very good precision [k combined equal to 0.91 (95% CI 0.87 - 0.95)]. In conclusion, this study highlighted a satisfactory reliability of most of the molecular methods used in Italy for DMV detection.
基金supported by the China-Australia Bilateral Research Program (No. 2010DFA31720)the National Key Technology R&D Program (2012BAD28B05)
文摘Noroviruses (NoVs) are widespread causes of nonbacterial gastroenteritis. Outbreaks of NoVs caused diseases are commonly ascribed to the consumption of contaminated shellfish. The concentration and RNA extraction of NoVs are crucial steps of detecting NoVs in shellfish. This study aimed to select a simple, rapid and highly efficient recovery method of NoVs detection with real-time RT-PCR. Four methods of recovering GI.3 and GII.4 NoVs from spiked digestive tissues of oysters and clams, respectively, were compared, of them, the method involving proteinase K and PEG 8000 was found the most efficient. With this method, 9.3% and 13.1% of GI.3 and GII.4 NoVs were recovered from oysters and 9.6% and 12.3% of GI.3 and GII.4 NoVs were recovered from clams, respectively. This method was further used to detect NoVs in 84 oysters (Crassostrea gigas) and 86 clams (Ruditapes philippinarum) collected from 10 coastal cities in China from Jan. 2011 to Feb. 2012. The NoVs isolation rates were 10.47% of clams (9/86) and 7.14% of oysters (6/84). All the detected NoVs belonged to genotype GII. The NoVs recovery method selected is efficient for NoVs detection in oysters and clams.