Use of broad range16S rDNA PCR in clinical microbiology

Sontakke, Sushama; Cadenas, María Belén; Maggi, Ricardo G.; Diniz, Pedro; Breitschwerdt, Edward B.

doi:10.1016/j.mimet.2008.11.002

Cited by 147 publications

(113 citation statements)

References 63 publications

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“…4,5 Pri 18-letni bolnici smo iz punktata vnetega kolenskega sklepa identificirali Neisseria meningitidis (možno je, da je šlo za septični artritis po asimptomatski meningokokcemiji brez simptomov), pri 24-letnem bolniku smo iz kolčnega sklepa dokazali Neisseria gonorrhoeae (Tabela 1). Pri tem bolniku je bila kultura sklepne tekočine ob prvem odvzemu negativna najverjetneje zaradi predolgega prenosa kužnine ob neustrezni temperaturi.…”

Section: Razpravljanjeunclassified

Evbakterijski PCR – uporabnost molekularne metode v klinični praksi

et al. 2015

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Background: Isolation and identification of bacterial pathogens enables accurate diagnosis of bacterial infection, allowing rational use of appropriate narrow-spectrum antibiotics. In some cases, the routine bacterial culture can give negative results. In those cases additional use of molecular techniques such as eubacterial (broad-range 16S rRNA) PCR may detect and identify bacterial genetic material.Methods: Between February 2012 and April 2013 42 specimens from 35 patients, already treated with antimicrobials, were taken and tested by eubacterial PCR in addition to routine microbiological culture. Results: Eubacterial PCR yielded positive result in 21/42 specimens in 18 patients (in three mixed sequences). Therefore, in 15 patients the diagnosis of bacterial infection was obtained with DNA identification and the results were interpreted in accordance to patients’ history, laboratory and image diagnostics. Only 4 specimens were culture-positive.Conclusions: Although eubacterial PCR enables the identification of any bacterial DNA in clinical specimens, there are some limitations: no information concerning antimicrobial susceptibility of the causative agents, problem of differentiating living from dead bacteria and problem to differentiate contaminants from pathogenic bacteria. The method is also expensive. In the following article recommendations for appropriate and rational use of eubacterial PCR are presented.

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Section: Razpravljanjeunclassified

Evbakterijski PCR – uporabnost molekularne metode v klinični praksi

et al. 2015

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“…Existen series amplias publicadas en la literatura en las que se ha demostrado su utilidad diagnóstica como, por ejemplo, en válvulas cardiacas, líquido cefalorraquídeo, tejido óseo y líquido articular 9 . El uso de esta metodología se restringe al análisis de muestras de sitio estéril, ya que la presencia de más de un agente dificulta la obtención de resultados concluyentes al secuenciar.…”

Section: A B O R a T O R I O C L í N I C O Pcr Universal O De Ampliunclassified

PCR universal o de amplio espectro: Un aporte a la detección e identificación de bacterias y hongos en la práctica clínica

Poggi

et al. 2009

Rev. méd. Chile

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“…The 16S rDNA polymerase chain reaction (PCR), based on the amplification of 16S rDNA in bacteria, is fast with high sensitivity and can fully detect the whole bacterial spectrum in the experimental samples (7). In combination with denaturing gradient gel electrophoresis (DGGE) and sequencing, 16S rDNA PCR has been used to detect the pathogens in the blood of patients suffering from fever or neutropenia (8).…”

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confidence: 99%

“…3A and B). When the E. faecalis DNA was amplified alone even at a 1x5 7 -fold dilution, the faint band amplified from E. faecalis DNA was still observed (Fig. 3A).…”

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16S rDNA PCR-DGGE and sequencing in the diagnosis of neonatal late-onset septicemia

Liu

et al. 2015

Molecular Medicine Reports

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Abstract. The 16S rDNA polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and sequencing method has been demonstrated to be valuable in detecting pathogens in the blood of patients suffering from fever or neutropenia. However, its use in the diagnosis of neonatal late-onset septicemia (LOS) has not yet been reported. The aim of the present study was to investigate the efficiency of this method in detection of the type of bacterial infection in neonatal LOS. Blood specimens from 60 neonates in whom LOS was suspected were collected. Fourteen culture positive blood samples and 24 spiked 'infected' blood samples were analyzed by the 16S rDNA PCR-DGGE and sequencing method or by pathogen-specific PCR. Only in 5 of the 14 cases did the results of 16S rDNA PCR-DGGE and sequencing match with the results of blood culturing. In the other 9 cases, the blood culture failed to detect bacteria, such as Neisseria sp. and Moraxella sp., which were detected by 16S rDNA PCR-DGGE and sequencing. Furthermore, the 16S rDNA PCR-DGGE and sequencing failed to detect blood culture-proven bacteria, such as Klebsiella pneumonia. A competitive inhibitory effect in 16S rDNA PCR amplification may lead to the discrepancy between pathogen-specific PCR and spiked 'infected' blood samples. When a certain species of bacteria was detected by 16S rDNA PCR, the competitive inhibitory effect presented a higher sensitivity in detecting this species in the blood samples that contained bacterial DNA only from this species compared with the blood samples that were blended with other bacterial DNAs. In conclusion, 16S rDNA PCR-DGGE and sequencing can detect a more comprehensive spectrum of pathogens than blood culture. However, the competitive inhibitory effect, which may lead to false negative results should be taken into consideration when the 16S rDNA PCR-DGGE and sequencing method is applied to the diagnosis of neonatal LOS.

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Use of broad range16S rDNA PCR in clinical microbiology

Cited by 147 publications

References 63 publications

Evbakterijski PCR – uporabnost molekularne metode v klinični praksi

Evbakterijski PCR – uporabnost molekularne metode v klinični praksi

PCR universal o de amplio espectro: Un aporte a la detección e identificación de bacterias y hongos en la práctica clínica

16S rDNA PCR-DGGE and sequencing in the diagnosis of neonatal late-onset septicemia

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