Identification Of Pathogenic Bacteria Causing Trauma Infection By Hybridization Analysis Of ITS Using Protable QCM Biosensor

BackgroundInfection is the most common complication of trauma patients, which is mainly caused by bacteria. It is reported that gram-negative bacillus are more than gram-positive coccus by the investigation of pathgenic bacteria flora causing trauma infection. In addition, multiple species of pathogenic bacteria causing mixed infection in one sample as well as increasingly different drug resistant strains are also be found. The advantages of rapid detection are self-evident since more rapid initiation of the correct antimicrobial therapy leads to a better outcome and less cost. Conventional bacteria culture method is generally reliable but time-consuming, laborious and expensive. Therefore, it is desirable to establish some rapid, simple and cost-effective diagnostic methods to detect pathogens causing trauma infection.At present, the use of molecular diagnostic techenique for bacteria identification has been a great advance for the rapid detection. As"living fossil"of bacteria, 16S rDNA is mostly selected to classification and identification from different bacteria. Sequencing or habridization analysis of the 16S rDNA for bacteria identification has been used in several studies. However, It has the disadvantage that it lacks the accuracy for discriminating genotypes within the same species and sometimes within the same genus. Comparatively, ITS not only consists of a series of conserved DNA stretches in all strains of a single species, but also includes more variable segments among different species. Therefore, the alternative of sequencing or habridization analysis of the ITS based on sequence and length variations should be more specific, which is similar to fingerprint identification. However, simultaneous identification of multiple pathogens present in the same sample is an important technical hurdle. QCM DNA biosensor, because of the inherent specificity of the principle of complementary base pairing, high sensitivity of physical transducers and convenient assembly of multiple array, has promising application to rapid, accurate and simultaneous identification of multiple clinical pathogenic microorganisms. We have set up a QCM DNA biosensor platform and successfully identified human papilloma virus, hepatitis B virus, staphylococcus epidermidis and human cytomegalovirus. However, the QCM DNA biosensor was applied to detect single pathogenic microorganism in those studies.In this study, We constructed a simple, cost-effective and portable 2×5 model QCM DNA biosensor array for rapid, simultaneous and accurate identification of five species of pathogenic bacteria causing trauma infection. Cl. perfringens, Cl. tetani and S. pneumoniae were especially selected because they were difficult to culture and identify, while P. aeruginosa and E. coli were the most common pathogens causing trauma infection. Using the self-designed universal primers, the ITSs of the bacteria were amplified by PCR and the PCR products were then analyzed by the QCM system. In the detection procedure, streptavidin conjugated nanoparticles were used as mass enhancers to amplify the frequency change, which could improve the detection limit and sensitivity. In addition, 50 clinical samples were detected by both the QCM system and conventional culture method to evaluate the performance of the QCM method in the pathogens identification and detection.Methods1. The universal primers were designed at bacterial conserved regions of the 3'end of 16S rDNA and the 5'end of 23S rDNA using Primer Premier 5.0 software. One or multiple short fragments including ITS of each bacterium were amplified by PCR. PCR products were examined using electrophoresis in agarose gel, and futher analyzed by sequencing. For a species that possessed multiple ITSs with different lengths and sequences, only the shortest fragment, usually the dominant band on the agarose gel, was eluted from the agarose gel and then sequenced.2. The biotinylated target DNA after hybridation was labeled with 5 nm gold nanoparticles to increase the mass load on the surface of quartz crystals by decreasing the response frequency of quartz crystals and further amplifying the frequency shift signals. The work concentration of gold nanoparticles was opitmized. Besides, the detection limit of synthesized oligonucleotide was determined.3. The PCR products of standard bacterial strains after purification and denaturation were detected by the QCM DNA biosensor using frequency shift signals amplification of gold nanoparticles. The respective probe was complementary to part of the shortest fragment, allowing it to hybridize with the PCR product. In addition, the specificity for detection of each bacterium and sensitivity for detection of P. aeruginos were tested.4. To demonstrate the use of rapid and simultaneous identification of 5 pathogenic bacteria causing trauma infection using portable QCM biosensor, 50 clinical samples including 31 cases of pus and 19 cases of wound secretion were detected individually using the QCM DNA biosensor system and the conventional bacteria culture method. The sensitivity and specificity of the current QCM DNA biosensor for bacteria detection of clinical samples were also analyzed.Results1. Confirmation of PCR amplification using gel electrophoresis and sequencing Using the universal primers, the ITS fragments of Cl. Perfringens, Cl. Tetani, S. pneumoniae, P. aeruginosa and E. coli were amplified by PCR without cross reaction to Human Genomic DNA, Candida Albicans or HBV DNA, which was highly specfic to bacteria. The PCR products were similar to what we expected on the agarose gel. The results of the sequencing and aligment showed that the identities were more than 99.5% for all bacteria, which confirmed bacterial ITSs could successfully be amplified by PCR with this method.2. The maximum frequency shift due to the amplification of gold nanoparticles was found at the final concentration of 9% (v/v). The threshold for the positive signal was set as background (blank) signal + 3×noise (standard deviation) and the detection limit was determined as 10?12 M. A linear relationship was found between the frequency shift and the logarithmic number of the positive control final concentration from 10?12 to 10?8 M, and the simple linear regression equation (y = 20.94lgx + 285.88, R2 = 0.958, P < 0.01) was obtained.3. Nonspecific frequency shift for each bacterium was significantly lower than that induced by a specific reaction (P < 0.05). These results indicate that this QCM system has relatively low cross-reactivity and acceptably high specificity. The threshold for the positive detection was set as before and the detection limit was determined as 1.5×102 CFU/ml, which was lower than the previously reported QCM DNA biosensor for bacteria detection. A linear relationship was found between the frequency shift and the logarithmic number of the cell concentration from 1.5×102 to 1.5×108 CFU/ml, and the simple linear regression equation (y = 12.921lgx ? 2.597, R2 = 0.975, P < 0.01) was obtained.4. Among 17 positive cases for 5 target bacteria diagnosed by the reference method using conventional bacteria culture techniques, 16 samples were correctly detected by the QCM DNA biosensor system. In addition, among 33 negative cases for 5 target bacteria diagnosed by the reference method, 3 samples were inconsistently detected by the QCM system. However, according to the McNemar test, there was no significant difference between the two methods (p > 0.05). Compared to conventional bacteria culture method, the diagnostic sensitivity of the QCM system assay resulted in 94.12 %, while the specificity resulted in 90.91 %.Conclusions1. Firstly, by using the self-designed universal primers, bacterial ITS which was more specific than 16S rDNA was successfully amplified by PCR, which lay a solid foundation for sequencing or habridization analysis.2. In addition, we constructed a simple, cost-effective and portable 2×5 model QCM DNA biosensor for rapid and simultaneous identification of five pathogenic bacteria which often causing trauma infection.3. Furthermore, The sensitivity of the system was significant enhanced by the gold nanoparticles and the detection limit was approximately 1.5×102 CFU/mL. This is a major improvement for the detection limit of the QCM biosensor for bacteria detection.4. Finally, the result of QCM biosensor is consistent well with the culture method. It is proved that the QCM biosensor can be applied to identify multiple pathegenic bacteria in clinical diagnosis, or even in the special environments including fields or natural disasters.