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A localized PCR inhibitor in a porcelain crab suggests a protective role.

El-Maklizi MA, Ouf A, Ferreira A, Hedar S, Cruz-Rivera E - PeerJ (2014)

Bottom Line: By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure.Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound.The identity of the inhibitory molecule remains unknown.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biology Department, The American University in Cairo , New Cairo , Egypt.

ABSTRACT
A number of polymerase chain reaction (PCR) inhibitors have been identified from biological and environmental samples. By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure. Here, we demonstrate the presence of a localized PCR inhibitor in the foregut of the porcelain crab Petrolisthes rufescens (Anomura: Porcellanidae) from the Red Sea. The inhibitor precluded amplification of 28s, 16s and 18s gene sequences effectively but lost activity at 10(-2) dilutions from initial concentration. Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound. The compound was not detected from midgut, hindgut, or gills of the crab. Activity of the inhibitor was precluded when samples were treated with suspensions from the midgut, suggesting that enzymatic degradation of the inhibitor likely happens at that part of the gut. As many microbial pathogens invade their hosts via ingestion, we suggest the presence of the localized inhibitor could carry a defensive or immunological role for P. rufescens. The identity of the inhibitory molecule remains unknown.

No MeSH data available.


Related in: MedlinePlus

Foregut inhibition of 16s amplification.Results of the amplification of bacterial DNA in crab tissues using universal 16s primers (see Methods). Samples from three different crabs were prepared to comprise either mixtures of midgut (MG), hindgut (HG) and gills (GL) together, or these three combined with foregut (FG) extracts from the same respective individual. The negative control assessed potential bacterial contamination and contained no crab or fish DNA. Empty lanes in the gel are not labeled.
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fig-3: Foregut inhibition of 16s amplification.Results of the amplification of bacterial DNA in crab tissues using universal 16s primers (see Methods). Samples from three different crabs were prepared to comprise either mixtures of midgut (MG), hindgut (HG) and gills (GL) together, or these three combined with foregut (FG) extracts from the same respective individual. The negative control assessed potential bacterial contamination and contained no crab or fish DNA. Empty lanes in the gel are not labeled.

Mentions: Despite yielding comparable amounts of DNA to control fish DNA concentrations, foreguts did not amplify when 28s primers were used, regardless of the amount of aliquot added to the PCR reactions (Fig. 1). In contrast, all five concentrations of control fish DNA showed amplified bands, with the best resolution when 2 µl of aliquot (ca. 2 ng) were added to the PCR reactions. Given the patterns of strong positive amplification of fish DNA in this experiment, 2 ng of fish DNA aliquots were used as controls to test for PCR inhibition in subsequent assays. Foregut aliquots ranging from 6–2 µl from different individual crabs arrested PCR in all cases (Fig. 2). In the absence of any foregut extract, 28s primers amplified fish DNA strongly and the negative control showed this amplification could not be explained by contamination of the mix (Fig. 2). This inhibition was seen when using bacterial 16s primers as well. When midgut, hindgut and gills from three crabs were extracted together and amplified using the bacterial primers, clear bands around 500 bp were observed (Fig. 3). In contrast, when the same mixtures from the same individuals also contained aliquots from the foregut, no bands were observed. A negative control showed no amplification either (Fig. 3). The inhibitor was effective up to one tenth of its concentration in the aliquots as shown in an experiment using 18s primers (Fig. 4). Further dilutions produced clear amplified product bands in the gels on the same expected positions as those from the positive controls (only fish DNA). No contamination of the mix (negative control) was detected (Fig. 4).


A localized PCR inhibitor in a porcelain crab suggests a protective role.

El-Maklizi MA, Ouf A, Ferreira A, Hedar S, Cruz-Rivera E - PeerJ (2014)

Foregut inhibition of 16s amplification.Results of the amplification of bacterial DNA in crab tissues using universal 16s primers (see Methods). Samples from three different crabs were prepared to comprise either mixtures of midgut (MG), hindgut (HG) and gills (GL) together, or these three combined with foregut (FG) extracts from the same respective individual. The negative control assessed potential bacterial contamination and contained no crab or fish DNA. Empty lanes in the gel are not labeled.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260131&req=5

fig-3: Foregut inhibition of 16s amplification.Results of the amplification of bacterial DNA in crab tissues using universal 16s primers (see Methods). Samples from three different crabs were prepared to comprise either mixtures of midgut (MG), hindgut (HG) and gills (GL) together, or these three combined with foregut (FG) extracts from the same respective individual. The negative control assessed potential bacterial contamination and contained no crab or fish DNA. Empty lanes in the gel are not labeled.
Mentions: Despite yielding comparable amounts of DNA to control fish DNA concentrations, foreguts did not amplify when 28s primers were used, regardless of the amount of aliquot added to the PCR reactions (Fig. 1). In contrast, all five concentrations of control fish DNA showed amplified bands, with the best resolution when 2 µl of aliquot (ca. 2 ng) were added to the PCR reactions. Given the patterns of strong positive amplification of fish DNA in this experiment, 2 ng of fish DNA aliquots were used as controls to test for PCR inhibition in subsequent assays. Foregut aliquots ranging from 6–2 µl from different individual crabs arrested PCR in all cases (Fig. 2). In the absence of any foregut extract, 28s primers amplified fish DNA strongly and the negative control showed this amplification could not be explained by contamination of the mix (Fig. 2). This inhibition was seen when using bacterial 16s primers as well. When midgut, hindgut and gills from three crabs were extracted together and amplified using the bacterial primers, clear bands around 500 bp were observed (Fig. 3). In contrast, when the same mixtures from the same individuals also contained aliquots from the foregut, no bands were observed. A negative control showed no amplification either (Fig. 3). The inhibitor was effective up to one tenth of its concentration in the aliquots as shown in an experiment using 18s primers (Fig. 4). Further dilutions produced clear amplified product bands in the gels on the same expected positions as those from the positive controls (only fish DNA). No contamination of the mix (negative control) was detected (Fig. 4).

Bottom Line: By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure.Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound.The identity of the inhibitory molecule remains unknown.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biology Department, The American University in Cairo , New Cairo , Egypt.

ABSTRACT
A number of polymerase chain reaction (PCR) inhibitors have been identified from biological and environmental samples. By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure. Here, we demonstrate the presence of a localized PCR inhibitor in the foregut of the porcelain crab Petrolisthes rufescens (Anomura: Porcellanidae) from the Red Sea. The inhibitor precluded amplification of 28s, 16s and 18s gene sequences effectively but lost activity at 10(-2) dilutions from initial concentration. Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound. The compound was not detected from midgut, hindgut, or gills of the crab. Activity of the inhibitor was precluded when samples were treated with suspensions from the midgut, suggesting that enzymatic degradation of the inhibitor likely happens at that part of the gut. As many microbial pathogens invade their hosts via ingestion, we suggest the presence of the localized inhibitor could carry a defensive or immunological role for P. rufescens. The identity of the inhibitory molecule remains unknown.

No MeSH data available.


Related in: MedlinePlus