外文翻译--亚硝酸盐扩增pcr引物系统的发展(编辑修改稿)

外文翻译--亚硝酸盐扩增pcr引物系统的发展(编辑修改稿)内容摘要：

ucts were visualized by an enzymelinked streptavidinbiotin coupling assay with a streptavidinalkaline phosphatase conjugate(GATC)and NBT/Xphosphate (Boehringer, Mannheim, Germany) as specified by the manufacturers. The sequences obtained were pared with published nirK and nirS sequences in the EMBL Nucleotide Sequence Database by FASTA analysis of the HUSAR program package based on the Geics Computer Group sequence analysis package . Hybridization analysis of nir products from total DNA of environmental samples. Approximately 100 ng (pure cultures) or 250 ng (environmental samples) of PCR product was analyzed on an agarose gel (2%, wt/vol). After electrophoresis, the DNA was transferred onto a positively charged nylon membrane samples. Approximately 100 ng (pure cultures) or 250 ng (environmental samples) of PCR product was analyzed on an agarose gel (2%, wt/vol). After electrophoresis, the DNA was transferred onto a positively charged nylon membrane (QIAbrane Nylon Plus。 Qiagen) by capillary transfer (24). The DNA was crosslinked to the membrane with UV light (45 s at 302 nm). Products generated with the primer bination nirK1FnirK5R from genomic DNA from Alcaligenes xylosoxidans NCIMB 11015 and with the bination nirS1FnirS6R from genomic DNA from Pseudomonas stutzeri ATCC 14405 were used as probes for nirK and nirS, respectively, to determine the specificity of nir products amplified from total environmental DNA. The nir products were purified by eluting the bands from an agarose gel by using the QIAquick gel extraction kit (Qiagen) as specified by the manufacturer. The probes were labeled randomly with digoxigenin by using the digoxigenin DNA labeling and detection kit (Boehringer) as specified by the manufacturer. The membrane was prehybridized in 20 ml of DIGEasy Hyb solution (Boehringer) for 2 h at 42176。 C. Hybridization was performed with 10 ml of DIGEasy Hyb solution containing the specific probe (25 ng ml21) and by incubation overnight at 42176。 C. After hybridization, the membrane was washed twice for 5 min at room temperature in 100 ml of a solution containing 23 SSC (13 SSC is M NaCl plus M sodium citrate) and % (wt/vol) SDS and twice at 45176。 C for nirK and 46176。 C for nirS for 15 min with 100 ml of a solution containing 3 SSC and % (wt/vol) SDS.. DISCUSSION The geic diversity of denitrifying bacteria in environmental samples can be investigated by different molecular methods. We describe herein the first steps required to detect denitrifying bacteria in aquatic habitats by the use of two distinct PCR systems for the nitrite reductase genes, nirK and nirS. Since denitrifiers are not defined by close phylogeic relationship, an approach involving 16S rRNA molecules is not suitable for general detection of this physiological group in the environment. The use of rRNAtargeted probes has been successfully applied so far only for strains and specific groups to explore the denitrifying munity of activated sludge . A more general approach to the detection of all denitrifying bacteria in environmental samples could be the use of a physiological gene or of an enzyme as a molecular marker. For this purpose, nitrite reductase and its genes have been used by several authors, since this is the key enzyme in the denitrification process. Antisera against the dissimilatory nitrite reductase (dNir) from Pseudomonas stutzeri ATCC 14405 were highly specific and reacted with the immunizing strain and few other closely related bacteria . Less specific reactions could be obtained with binations of antisera against hemetype dNirs from P. stutzeri JM300 and P. aeruginosa . When a variety of approximately 150 denitrifying strains of uncharacterized dNir type were screened with this bination and an antiserum against Cu dNir from Alcaligenes cycloclastes, 90% of the strains could be identified as possessing either the hemetype or Cu dNir . Due to the inducible nature of the enzyme, antisera could be useful in detecting conditions of active denitrification . In contrast, approaches targeting the nirK or nirS gene would detect the denitrifiers irrespective of the denitrifying conditions. Compared to the antisera, a broader response to different pure cultures possessing the nirS gene was achieved by use of the specific gene probes (15, 24). By using hybridization with a gene probe for nirK, this type of nitrite reductase could be always confirmed in pure cultures . In enrichment cultures, different populations of denitrifiers could be detected by restriction enzymedigested preparations (HindIII) of total DNA with a probe for nirS . PCR amplification systems, on the other hand, are limited neither to actively denitrifying cells nor to cultivated strains. Application to pure cultures of a primer pair derived from three nirS sequences flanking a conserved central region of the nirS gene revealed a reactivity broader than that with the use of antisera but not as satisfactory as that with the use of gene probes . A strainspecific reaction such as that obtained with the use of antisera was not achieved, although amplification failed with some nirScontaining strains as detected by hybridization. This may be because for PCR amplification only the homology of the primer hybridization region is decisive whereas hybridization of gene probes can be detected if any region of the probe shows sufficient homology. In the present study, a PCR system based on six sequences each for nirK or nirS available from the database promised an even mor。