Background:Burkholderia bacteria are species from protobacter and the pathogens of this species include Burkholderia mallei and pseudomallei causing glanders. The yellow fever virus is a blavovirus that has a single-stranded polyribonucleotide genome (positive strand). Yellow fever is a type of acute viral disease, also called black vomit. Even though considerable advances have been made in the development and use of molecular techniques for the detection of pathogens such as Burkholderia species and yellow fever, a major problem in these techniques is lack of proper positive control. Recently developed genetically modified vectors and simulator construct have provided engineered tools that can be used as a positive control in molecular diagnosis.
Methods:In the current study, we first designed and produced chimeric simulator constructs, containing specific genes from Burkholderia mallei, pseudomallei, and yellow fever virus. Then, the operational efficiency of the designed structures was evaluated using a single and dual-mode quantitative (using SYBER Green) and qualitative (using the TaqMan probe) Real-time PCR.
Results:The minimum and maximum sensitivity of designed construct for Burkholderia and yellow fever detection was indicated to be 0.1 pg and 10 ng, respectively.
Conclusions:Our findings suggest that this positive control constructs can be used for development of new diagnostic kits.
Burkholderia is a genus of Proteobacteria with pathogenic members including the Burkholderia cepacia complex (a human pathogen) and Burkholderia mallei (responsible for glanders in horses and related animals) (1, 2). They are gram negative, aerobically and bar-shaped, which can develop resistance to antibiotics. One of the essential steps in detection of the pathogens (such as Burkholderia mallei and pseudomallei) is culturing them in microbial standard culture media (2).
The yellow fever virus (YFV) is a mosquito-borne flavivirus that is the causative agent of yellow fever (also called black vomit). Its genome is a single-stranded RNA of positive polarity that is ∼11 kb in length (3, 4). Yellow fever is an anthropoid zoonosis disease (5). The disease is common in tropical regions of South America and Africa, but is not common in Asia. Yellow fever is difficult to diagnose and can be confused with malaria, typhoid and other haemorrhagic fevers such as dengue (6).
Traditional methods for detection of pathogens bacteria are based on isolation of bacteria from patient’s body, culturing them and performing bacteriological diagnostic test. However, development of PCR-based diagnostic methods, which are faster and more accurate, became widespread rapidly (7-11). Currently, real-time PCR technique is a gold standard method used in diagnosis of vast number of pathogens. This highly specific, sensitive and repeatable technique, gives us the possibility to detect even vary small amount of the target in a sample (10, 11). However, this method requires direct intervention of technical operator with pathogens, increasing the risk of infection for the operator. Thus, development of constructs that have specific features of targeted pathogens, while lacking pathogenic properties, would provide a safe and reliable calibrated control for pathogen detection.
Plasmids were the first construction to be used as positive controls in amplification reactions for Burkholderia species detection (12). Vectors (or plasmids) are naturally occurring small pieces of circular DNA that can replicate in various organisms. They are engineered to transfer specific genetic sequences into target tissues (13). This special ability to carry the genetic component makes them ideal candidate to be used as control constructs, minimizing the problems such as absence of an appropriate positive genetic control for certain microbial and viral agents, occurrence of false positive results and unavailability of commercial tests for some pathogens (12, 14, 15).
In current study, a positive control was designed and synthesized to detect, Burkholderia mallei, psudomallei and yellow fever. By using respective primers and probes, detection of construct was evaluated. By quantitative (using SYBER Green color) and qualitative (using a TaqMan probe) real-time PCR, the operational efficiency of the designed structure and the sensitivity and specificity of the primers and probes were evaluated.
3.1. Steps in Designing and Production of Genetically Simulator Construct Primers and Probes
A: all nucleotide sequences from each of the targets, including Burkholderia mallei and psudomallei and yellow fever virus, were downloaded as FASTA files from International Nucleotide Sequence Database Collaboration (INSDC).
Then all the nucleotide outputs of the BLAST-N test at the NCBI site (with the selection of the NR database) for selected target sequences (mostly containing 100 sequences) were collected in a FASTA file. As these sequences are very long, multiple alignments are sometime difficult. Therefore, we tried to extract short sequences (mostly under 2 kb) for selected genes from the nucleotide sequence of a specific pathogen, so that in cases where the volume and length of sequences is high, this new file in the multiple alignment process was used.
In the next step, conserved sequences within genome of these pathogens were identified by using at least 3 software for sequence analysis and multiple alignment process (including MEGA, AliView and BioEdit, Clustal W).
Finally, a sequence of conserved region with 177 bp length of the Burkholderia mallei and psudomallei species and a conserved sequence with 226 bp length for yellow fever virus was used to produce a simulator construct. These sequences were cloned within the plasmid pUC57 and transferred into E.coli DH5α. Plasmids were allowed to replicate overnight by incubating transfected E.coli DH5α at 37°C in a LB culture medium. Then the plasmids were extracted with plasmid extraction kit (GeneAll®ExprepTM Plasmid SV mini) and collected plasmids were used for next steps.
B: at the next step specific changes in the target gene region for insertion into the simulator construct was introduced. After designing the fully customized primers for the specific and conserved areas, specific sequence was added to or removed from middle region of the Burkholderia and yellow fever target sequences in simulator construct. This changes causes these sequences to become shorter or longer in the construct, compared to original sequence in above mentioned agents. This aids us to distinguish target sequences in simulator construct from original sequence. Furthermore, restriction enzymes sites, EcoRI and NotI (EcoRI -GAATTC- and NotI -GCGGCCGC-), were inserted into the target sequences of simulator construct. After cutting the PCR product by these two restriction enzymes the PCR products of the simulator construct and the genome of suspected sample were separated on the gel to make 2 independent fragments. Besides, by applying these enzymes on a suspect sample, prior to PCR reaction, we ensure the removal of artificial simulator constructs which may exist due to laboratory contamination in suspected sample. In fact, these steps reduce the error rate and eliminating false positive results (Figure 1).
C: probes and primers were designed by AllelID designing software based on the 16S ribosomal RNA gene sequence, coded by the Locus MH006571 gene (part of the conserved area), for the Burkholderia mallei and pseudomallei species and the whole genome for yellow fever (Table 1).
|Target Organism||Target Sequence||The Sequence of Primers and Probes Desired||Length||Real Sequence Size|
|Burkholderia mallei and pseudomallei||16S ribosomalRNA gene||Forward primer||TTCTGGCTAATACCCGGACTG||21||177 bp|
|Yellow fever||Whole genome||Forward primer||ACCCACACATGCAGGACA||18||226 bp|
|Probe||JOE -TCCGGATGCGGTGTATTACCAAGTGGAT- BHQ2||28|
3.2. Performing a Single-Mode Real-Time PCR Test
3.2.1. Quantitative PCR by SYBR Green
Real-time PCR reaction was performed according to manufactures instruction. Briefly, the mixture was prepared by adding 5 μL of SYBR Green, 0.5 μL of the forward and reverse primer mixture (5 pM), the variable amount (serially from concentrations of 100, 10 and 1 ng/μL) of the plasmid pUC57 up to 10 μL of distilled water for each sample in a sterile microtube. After adding the mix to the Strip Wells, the dilutions of the plasmid pUC57 were added and the strips were covered with a special cover. PCR reaction was performed with following setting: one cycle, for 15 minutes, at 95°C ,and 40 cycles, denaturation: 5 seconds, at 95°C, annealing: 25 seconds, at 62°C, extension: 30 seconds, at 72°C
3.2.2. Quantitative Analysis Using TaqMan Probe
The PCR mixture was prepared by adding 5 μL of TaqMan master mix, 0.5 μL of the forward and reverse primer (5 pM), 0.5 μL of probe, the variable amount (Serially at concentrations of 100, 10, and 1 ng /μL, 100, 10, and 1 pg /μL, 100, 10, and 1 Fmtg/μL) to a volume of 10 microliter in a sterile microtube. After adding the mix to the strips wells, various dilutions of the plasmid pUC57 were added and the strips were covered with a special cover. For PCR, the predenaturation program: one cycle, 10 minutes, at 95°C, and 40 cycles, denaturation: 5 seconds, at 95°C, annealing/extension: 30 seconds, at 62°C were designed. Finally, products were transferred to 1.5% agarose gel and the resulting images were stored.
3.2.3. Performing Multiple Real-Time PCR
Five μL of TaqMan mastermix, 0.5 μL of the forward and reverse primer (5 pM) of Burkholderia mallei and psudomallei and yellow fever virus, 0.5 μL of the probe (5 pM) Burkholderia mallei and psudomallei and yellow fever, variable amount of pUC57 plasmid (serial concentrations of 100, 10, and 1 ng/μL, 100, 10, and 1 pg/μL, 100, 10, and 1 Fmtg/μL) and up to 10 μL of distilled water were mixed. After adding the mixture to the strips wells, the dilutions of the plasmid pUC57 were added and the strips were covered with a special cover. Then, placed inside the Corbett Rotorgen 6000 machine and centrifuged at 1000 rpm. For PCR, the program was set as followed: one cycle, 10 minutes, at 95°C, and 40 cycles, denaturation: 5 seconds, at 95°C, annealing/extension: 30 seconds, at 62°C. Finally, amplification products were transferred to 1.5% agarose gel and the resulting images were analyzed.
3.3. Statistical Analysis
The statistical analysis was performed on at least three replicates, which was calculated by taking the mean and calculating the standard deviation. The nonparametric Wilcoxon method was used to compare the mean of the groups.
4.1. Single Real-Time PCR
A: real-time PCR assay was performed using SYBR Green by various dilutions of 100, 10, 10, 1, and 0.1 ng of the plasmid (2 replicates). The observed amplification curves for plasmid serial dilution was correlated with its concentration; i.e., the more plasmid, led to lower the threshold cycle (Ct) value. Besides a single melting curves was observed, indicating the specific and individual amplification of the corresponding product on the simulator construct (Figure 2).
B: real-time PCR test was performed using TaqMan Probe at 100 ng, 1 ng, 100 pg, 10 pg and 1 pg of plasmid (with 2 replicates). Amplification curves by various plasmid dilutions showed that as its concentration increased, the threshold cycle (Ct) values were reduced. Also, the single fragment of the product was observed on the gel, indicating specific and individual amplification of the corresponding product on the simulator construct (Figure 3).
4.2. Performing a Multiplex Real-Time PCR
Multiplex real-time PCR test was performed using TaqMan probe at 100 ng, 1 ng, 100 pg, 10 pg, and 1 pg of plasmid (with 2 replicates). For differentiating amplification curve of Burkholderia from yellow fever, Burkholderia probe had FAM fluorophore and yellow fever probe had JOE fluorophore. Amplification curves for Burkholderia and yellow fever were observed in FAM channel and in JOE channel respectively. Amplification curves for plasmid serial dilution showed that by increasing the relative dilution of the plasmid, the Ct values were reduced. A single band of the product on the gel was observed indicating specific and individual amplification of the corresponding product on the simulator construct (Figures 4 and 5).
4.3. Sensitivity and Precision
As studies on simulator constructs have been conducted rarely and available studies are performed on target bacterium or its genome, not the artificial structure, we could not compare our data with others.
However, the minimum and maximum sensitivity of previous studies for detection of bacteria of Burkholderia and yellow fever was reported to be 0.01 pg and 1 μg for Burkholderia and 1 pg and 10 ng for yellow fever (16-20). This values were demonstrated to be 0.1 pg and 10 ng with the construct designed for both agents, in our study.
In current study we have designed an artificial construct as a novel and reliable method for simultaneous detection of Burkholderia species and yellow fever virus. It is also possible to avoid or to ensure immediate detection of false positive results due to contamination by positive controls using these plasmids.
Currently available molecular diagnosis techniques for the detection and identification of highly infectious agents require direct encounter of technical operator with pathogen. The process should be performed only in clinical laboratories that have specific equipment and facilities.
Besides, safety regulations, obtaining nucleic acids content of potential pathogen for calibration and application as a positive control is difficult. Moreover, if the multiple microorganisms are being tested simultaneously various controls are required. Besides, incidence of false positive results due to contamination from positive controls is possible. In our study we proposed a construct to be used as safe positive control for detection of Burkholderia mallei and pseudomallei as well as yellow fever virus. Such constructs were proposed by Charrel et al. (12) for detection of highly dangerous agents. By introducing the sequences of interest into the plasmid and detecting the sequence by primers and probes they demonstrated highly sensitive and applicable method for pathogen detection.
In other study by Sagripanti and Carrera, a single conserved sequence from all Burkholderia species was used in construct. By inserting a specific polymorphism for Burkholderia mallei, into this construct it was able to distinguish it from other bacteria as well as other genus Burkholderia (14). Our design construct was able to detect Burkholderia mallei and psudomallei, as well as yellow fever virus simultaneously. We suggest that this construct provides safe, non-infectious chimeras mimicking properties of pathogenic agents, without being pathogen.
Development of simulants to be applied in molecular diagnostic techniques provide a useful tool as nucleic acid-based biodetectors for diagnostic approaches, this method does not require accessing or producing treat agents in laboratory. However, as mentioned earlier, even though this approach is promising, there is not much research for the production of such simulator constructs.
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