Development and evaluation of a quantitative polymerase chain reaction for aquatic
            <i>Streptococcus agalactiae</i>
            based on the
            <i>groEL</i>
            gene

Leigh, William J.; Zadoks, Ruth N.; Costa, Janina Z.; Jaglarz, Anita; Thompson, Kim D.

doi:10.1111/jam.14556

Cited by 10 publications

(11 citation statements)

References 28 publications

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“…PCR mixtures were comprised of 10 μl 2X SYBR Green Master Mix (Bio-Rad), 0.5 μl of each forward and reverse primers (10 μM of each TiPV-Fq/TiPV-Rq) (Table 1), 2 μl DNA template and 7 μl ddH 2 O. PCR was performed at 94°C for 2 min followed by 40 cycles of 94°C for 10 s and 60°C for 30 s. The melting curve was analysed in the last cycle. On the other hand, probe-based qPCR targeting groEL gene was used to diagnose S. agalactiae infection, according to Leigh et al (2020). The qPCR reagents mixture contained 10 μl of 2X iTaq Universal Probes Supermix (Bio-Rad), 0.5 μl of 10 μM SagroEL-probe (Table 1), 1.8 μl of each primer SagroEL-F/R (10 μM), 3 μl of DNA template and 2.9 μl ddH 2 O.…”

Section: Molecular Detection Of Tipv and S Agalactiaementioning

confidence: 99%

Usefulness of the pancreas as a prime target for histopathological diagnosis of Tilapia parvovirus (TiPV) infection in Nile tilapia, Oreochromis niloticus

Dong

Sangpo

Dien

et al. 2022

Journal of Fish Diseases

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Tilapia parvovirus (TiPV) is an emerging virus reportedly associated with disease and mortality in farmed tilapia. Although previous descriptions of histopathological changes are available, the lesions reported in these are not pathognomonic. Here, we report Cowdry type A inclusion bodies (CAIB) in the pancreas as a diagnostic histopathological feature found in adult Nile tilapia naturally infected with TiPV. This type of inclusion body has been well-known as a histopathological landmark for the diagnosis of other parvoviral infections in shrimp and terrestrial species. Interestingly, this lesion could be exclusively observed in pancreatic acinar cells, both in the hepatopancreas and pancreatic tissue along the intestine. In situ hybridization (ISH) using a TiPV-specific probe revealed the intranuclear presence of TiPV DNA in multiple tissues, including the liver, pancreas, kidney, spleen, gills and the membrane of oocytes in the ovary. These findings suggest that although TiPV can replicate in several tissue types, CAIB manifest exclusively in pancreatic tissues. In addition to TiPV, most diseased fish were co-infected with Streptococcus agalactiae, and presented with multifocal granulomas secondary to this bacterial infection. Partial genome amplification of TiPV was successful and revealed high nucleotide identity (>99%) to previously reported isolates. In summary, this study highlights the usefulness of pancreatic tissue as a prime target for histopathological diagnosis of TiPV in diseased Nile tilapia. This pattern may be critical when determining the presence of TiPV infection in new geographic areas, where ancillary testing may not be available. TiPV pathogenesis in this landmark organ warrants further investigation.

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Section: Molecular Detection Of Tipv and S Agalactiaementioning

confidence: 99%

Usefulness of the pancreas as a prime target for histopathological diagnosis of Tilapia parvovirus (TiPV) infection in Nile tilapia, Oreochromis niloticus

Dong

Sangpo

Dien

et al. 2022

Journal of Fish Diseases

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“…For the molecular methods, standard PCR for 16S rRNA gene yielded the lower sensitivity for S. agalactiae DNA presence. The overall percentage of positive results, similar to a culture technique, reached 17.2% and was relatively low, when As mentioned previously, the choice of a particular gene and the primers or protocol parameters is crucial for the reliable results of real-time PCR-based results [2,18,19]. For example, molecular methods may be improved if they are based on consensus sequences from a relatively high number of GBS genomes, including those characteristic for particular bacteria and their conserved genes, such as the sip or cfb genes for S. agalactiae.…”

Section: Discussionmentioning

confidence: 99%

Application of the appropriate molecular biology-based method significantly increases the sensitivity of group B streptococcus detection results

Bogiel

Depka

Zalas‐Więcek

et al. 2021

Journal of Hospital Infection

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Background: Streptococcus agalactiae (group B streptococcus: GBS) is a leading cause of early-and late-onset diseases in neonates. Reliable results of GBS carriage investigation among pregnant women may decrease the incidence of neonatal infection and mortality. Aim: To compare the results of conventional culture investigation with those of the US Food and Drug Administration-approved nucleic acid amplification test (BD Max GBS (Becton Dickinson)), and to establish our own protocols of standard polymerase chain reaction (PCR). Methods: A total of 250 vaginalerectal swabs from three different hospitals in Bydgoszcz, Poland, were used to evaluate GBS carriage. Standard laboratory technique (overnight culture in broth enrichment media) results were compared with those of BD Max GBS assay (Becton Dickinson) and two standard PCR protocols, established to detect the cfb and 16S rRNA S. agalactiae genes, from the overnight cultures of the samples in the liquid enrichment media. Findings: The overall GBS carriage was estimated as 16.4e23.2%, depending on the applied detection method. The highest percentage of positive results, from each laboratory was obtained with the application of BD Max GBS assay. The differences in the number of positive results obtained with this particular method were statistically significant. Overall, 27 discrepancies were noted for the results obtained with the application of the methods compared. Conclusions: The methods applied for GBS detection differ in sensitivity. A culture technique, though very specific, appears to be less sensitive at detecting S. agalactiae compared with the commercially available BD Max GBS assay or in-house PCR protocols established for this purpose.

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“…PCR mixtures were comprised of 10 μL 2X SYBR Green Master Mix (Bio-Rad, USA), 0.5 μL of each forward and reverse primers (10 μM of each TiPV-Fq/TiPV-Rq) (Table 1), 2 μL DNA template and 7 μL ddH 2 O. PCR was performed at 94 °C for 2 min followed by 40 cycles of 94 °C for 10 s and 60 °C for 30 s. The melting curve was analyzed in the last cycle. On the other hand, probe-based qPCR targeting groEL gene was used to diagnose S. agalactiae infection, according to Leigh et al (2019). The qPCR reagents mixture contained 10 μL of 2X iTaq Universal Probes Supermix (Bio-Rad, USA), 0.5 μL of 10 μM SagroEL-probe (Table 1), 1.8 μL of each primer SagroEL-F/R (10 μM), 3 μl of DNA template and 2.9 μL ddH 2 O.…”

Section: Methodsmentioning

confidence: 99%

Usefulness of the pancreas as a prime target for histopathological diagnosis of Tilapia parvovirus (TiPV) infection in Nile tilapia, Oreochromis niloticus

Dong

Sangpo

Dien

et al. 2022

Preprint

View full text Add to dashboard Cite

Tilapia parvovirus (TiPV) is an emerging virus reportedly associated with disease and mortality in farmed tilapia. Although previous descriptions of histopathological changes are available, the lesions reported in these are not pathognomonic. Here, we report Cowdry type A inclusion bodies (CAIB) in the pancreas as a diagnostic histopathological feature found in adult Nile tilapia naturally infected with TiPV. This type of inclusion body has been well-known as a histopathological landmark for the diagnosis of other parvoviral infections in shrimp and terrestrial species. Interestingly, this lesion could be exclusively observed in pancreatic acinar cells, both in the hepatopancreas and pancreatic tissue along the intestine. In situ hybridization (ISH) using a TiPV-specific probe revealed the intranuclear presence of TiPV DNA in multiple tissues, including the liver, pancreas, kidney, spleen, gills, and the membrane of oocytes in the ovary. These findings suggest that although TiPV can replicate in several tissue types, CAIB manifest exclusively in pancreatic tissues. In addition to TiPV, most diseased fish were co-infected with Streptococcus agalactiae, and presented with multifocal granulomas secondary to this bacterial infection. Partial genome amplification of TiPV was successful and revealed high nucleotide identity (> 99%) to previously reported isolates. In summary, this study highlights the usefulness of pancreatic tissue as a prime target for histopathological diagnosis of TiPV in diseased Nile tilapia. This pattern may be critical when determining the presence of TiPV infection in new geographic areas, where ancillary testing may not be available. TiPV pathogenesis in this landmark organ warrants further investigation.

show abstract

Development and evaluation of a quantitative polymerase chain reaction for aquatic Streptococcus agalactiae based on the groEL gene

Cited by 10 publications

References 28 publications

Usefulness of the pancreas as a prime target for histopathological diagnosis of Tilapia parvovirus (TiPV) infection in Nile tilapia, Oreochromis niloticus

Usefulness of the pancreas as a prime target for histopathological diagnosis of Tilapia parvovirus (TiPV) infection in Nile tilapia, Oreochromis niloticus

Application of the appropriate molecular biology-based method significantly increases the sensitivity of group B streptococcus detection results

Usefulness of the pancreas as a prime target for histopathological diagnosis of Tilapia parvovirus (TiPV) infection in Nile tilapia, Oreochromis niloticus

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