Throughout the world, blood stream infections (BSIs) show high rates of morbidity and mortality, with a mortality rate ranging from 20 to 70%. ¹,²,³,⁴,⁵ BSIs account for 30-40% of all cases of severs sepsis and septic shock.- ⁶ The true incidence of nosocomial BSI is unknown, but it is anticipated that about 250,000 cases occur annually in the USA. ⁷ Affirmation of the septicemia diagnosis depends on microbiological exams based on blood culture procedure, which usually takes 24-72 h. ⁸ Thus, in many cases, antibiotic therapy is initiated on the basis of clinical criteria. Inappropriate empirical antimicrobial therapy is an important predictor of death, especially among patients in critical condition, and is associated with the emergence of drug-resistant strains, increasing the treatment costs. ⁹ Nevertheless, it is suggested that adequate antimicrobial therapy should be initiated as early as possible because a delay by each hour results in a 7.6% increase in death rate. ¹⁰

Faster diagnosis of BSIs by molecular assay using polymerase chain reaction (PCR) is an important tool for the detection of microorganisms with high sensitivity, even when targets are present at extremely low number [10-100 deoxyribo nucleic acid (DNA) copies]. ¹¹ Earlier implementation of appropriate antimicrobial treatment reduces morbidity and mortality. ¹² Enterobacteriaceae is the most common gram-negative bacteria causing septicemia and there is approximately the same remediation protocol for them according to clinical and laboratory standards institute (CLSI). ¹³ Microbial investigation by PCR has been explored with a detection system known as multiplex PCR. This technique is based on simultaneous amplification of two or more primer pairs in a single reaction vessel, which, in turn, usually means reduced cost and time requirement. The multiplex PCR technique requires a complex balance among some parameters such as level of salts, primers, DNA polymerase, and compatible primer melting temperatures, resulting in a laborious experimental design. ¹⁴,¹⁵ One of the most important factors in PCR assay is checking the reaction by negative and positive controls. ¹⁶

The aim of present study was to develop and standardize a set of primers and to evaluate the analytical sensitivity and specificity of specific DNA fragments of bacteria and internal control, which is controlled the whole of the process is needed for assay such as DNA extraction and PCR reaction, with a simple, fast, and cost-effective method. There isn′t any stydy, which used rpoB gene and internal control (IC) in the same reaction to identification of enterobacteriaceae in bacteremia by PCR.

This study is a cross-sectional kind that was performed in Isfahan University of Medical Science, Isfahan, Iran, in 2011-2012 as part of a research project number 390011. Reference strains of organisms were bought from reference laboratory of Iran and Pasture Institute of Iran and brought to the microbiology lab of bacterial and virology group, Medical School, Isfahan University of Medical Science.

Primer design

A panel of six primers was developed for detection of enterobacteriaceae organisms related to sepsis, 16S rDNA, and IC. A series of nucleotide sequence of each organism was selected from the GenBank database returned from the rpoB gene and 16S rDNA for all of the microorganisms and X chromosome of Drosophila melanogaster for IC. Each respective set of consensual sequence was generated by Mega4 clustalW software. ¹⁷ The sequences were submitted to Allel ID6 software to design a primer for template. Then, the designed primer was checked in Mega4, Oligo6, Oligo Explorer software for finding the sequence and annual temperature, and simulating a set of primers for simultaneous amplification, respectively. The primer sets and genes target sequences are listed in Table 1.{Table 1}

Multiple oligonucleotides were then tested in silicon regarding specificity of the BLAST tool (BLAST N and Primer BLAST) against all nucleotide sequences available in GenBank.

Microorganisms

Reference strains of organisms such as Escherichia coli (ATCC 25922), Enterobacter aerogenes (ATCC 13044), Proteus mirabilis (ATCC 7002), Klebsiella pneumonia (ATCC 13882), Salmonella enterica (ATCC 35640), and Shigella boyidii (ATCC 9207) were bought from reference laboratory of Iran and Pasture Institute of Iran.

DNA preparation

We used six different bacterial strains such as: Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213 as Gram positive and Acinetobacter baumannii ATCC 19606, E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and K. pneumonia ATCC 13882 as Gram negative using three different kits: a-Qiamp DNA Mini Kit (Qiagen, Germany ), b-Fermentas (Fermentas, Lithuania), and c-Cinagen DNA extraction kit (dNTP, Iran) to evaluate efficiency was applied for DNA extraction of strains maintained in solid media as follows: 200 μl of bacterial suspension (0.5 McFarland) was subjected to individual extraction protocols as indicated by each manufacturer. Qualitative and quantitative assessment of DNA preparation was performed through agarose gel and spectrophotometric measurements.

Bacteria and D. melanogaster (IC) DNA quantitation

The linearized standard PTZ75 plasmid was used for generation of a standard. The standards of 16S rDNA, K. pneumonia, and IC were made and measured according to the Applied Biosystems′ protocol. Then, sensitivity tests were performed by TA cloning assay (Fermentas, Lithuania) according to manufacturer′s instructions, and colony PCR of mentioned microbial strains and IC was carried out. The obtained band of each dilution is plotted against the log10 of the corresponding initial template concentration. It was done by testing eight different dilutions, each one in triplicate, for 3 days by PCR. The concentration was 1 × 10 ⁸ to 1 plasmid DNA target sequences copy number/reaction and repeat for the others.

Polymerase chain reaction

The reaction was performed at a defined volume of 25 μl and included 50-500 ng DNA, 12.5 μl Master Mix (Takar a, Japan), and 0.5 μl of 10 pM primer (Faza Biotech, Iran). To achieve a common optimal annealing temperature for all primer pairs, individual reactions were performed at 60°C. The initial denaturation was performed at 94°C for 5 min, followed by 35 cycles encompassing a 30-second step at 94°C, a 30-second step at 60°C, and a 1-min step at 72°C. An extension step at 72°C for 10 min ended the procedure. The amplicons obtained were submitted to electrophoresis in 1% agarose gel and stained by ethidium bromide (0.5 μg/ml) for UV light analysis and digitized (UV doc).

Analytical sensitivity and specificity of the test

Sensitivity was tested with serial dilutions of different plasmids containing the target sequences of the 16S rDNA, K. pneumonia, and IC, ranging from 1 to 10 ⁸ copies/reaction. Specificity tests were performed employing pure cultures DNA of microorganisms included or not included in the panel such as: A. baumannii (ATCC 19606), K. pneumonia (ATCC 13882), P. aeruginosa (ATCC 27853), E. faecalis (ATCC 29212), S. aureus (ATCC 29213), and human DNA.

Statistical analysis

The one-way analysis of variance (ANOVA) test was used to compare the three different extraction kits. P values below 0.05 were considered significant.

Analysis of multiple oligonucleotide sequences suggested by Oligo Analyzer software resulted in a panel of three pairs of compatible primers for amplification of respective microorganisms in the same protocol. The 16S rDNA primers were designed by degenerative technology for amplification of all the bacteria and generated amplification of 1505 bp, the IC generated amplification of 684 bp, and K. pneumonia generated amplification of 512 bp Table 1. Analysis of all primers was carried out at an annealing temperature of 60°C. Multiplex PCR was performed with K. pneumonia, IC, and 16S rDNA Figure 1.Figure 1

Simultaneous amplification in one vessel of Klebsiella pneumonia, Internal control (spiked IC) and 16S rDNA with optimized common annealing temperature (60° C) and specificity test of the PCR system designed for: (1) K. pneumonia (2) S. aureus (3) E. facium (4) P. aeruginosa (5) A. baumani (6) Human DNA (7) H₂O. The bacterial concentration is (0.5 McFarland) of each strain. (M) Molecular Standard is indicated on the right

Diagnostic procedures for sepsis require a 24-72 h period for confirmation of an infectious etiology and identification of the pathogen, and antibiotic therapy is usually started on an empirical basis. ⁸ However, the survey of comparing blood cultures and another technique for detection of microorganisms related to sepsis involves more rapid and accurate reporting when performed using PCR-based methods. ^{[18 ],19,[20 0]} DNA isolation and amplification is very important for almost all laboratories involved in molecular biology research of microorganisms, especially those dedicated to difficult samples. Purity and quality of this DNA has to be verified before doing any amplification reactions; therefore, there is an increasing need for new, fast, and reliable method. ²¹

The time required for specific enterobacteriaceae diagnosis using the multiplex PCR developed in the present study is about 4 h. It must be considered that this panel includes the detection of IC for confirming DNA extraction and PCR reaction in view of the difficulty of samples. Considering the high sensitivity of PCR-based methods, specific measures must be followed to prevent false-positive results; such measures include sequencing using specific primers for confirming the obtained amplicons. ²² Our results showed a detection limit variable between 1 and 100 copies of the target sequences when they were used separately, but in multiplex PCR, the detection limit is 10 times lower than this. Similar detection limits were observed for different molecular techniques when applied to different samples.- ²³,²⁴ On the other hand, we used IC to prevent false-negative results. Other studies have proposed variety methods of PCR-based diagnostic procedures. ^{[25 ],26,[20 7]} For example, a molecular method could detect 62 pathogens via PCR followed by hybridization assay; ²⁸ however, this assay does not include microorganisms′ differentiation. In the present study, a set of primers was reported for detecting the most common gram-negative bacteria included in enterobacteriaceae which involve 30-40% of microorganisms related to sepsis. It is a faster method and showed higher sensitivity than blood culture. In the other hand, there was an IC for detecting the whole of DNA extraction and PCR reaction process, which made ensure of the result especially about negative ones. Furthermore, fast and sensitive molecular panel-based method for detecting enterobacteriaceae causing sepsis directly from blood specimen appears promising for a rapid fine tuning of empirical antibiotic therapy. ²⁰

In comparison to the multiple conventional methods where all primers are mixed in a single vessel, there is no delay in diagnostic time. However, there is a 16S primer accompanying an IC, which confirmed the existence of bacteria and accuracy of all steps of PCR process. We conclude that the molecular method developed is a reliable and fast method for detecting sepsis related to enterobacteriaceae; however, the role of this method in standard strains was assessed and its sensitivity and specificity in analytical step was measured, but further studies must be undertaken on clinical samples to confirm its application in diagnostic laboratory.

The authors are grateful to Vice Chancellor for Research of the Isfahan University of Medical Sciences for providing financial support for this study.