Simultaneous Amplification Research Articles

Two-frequency amplification in a semiconductor tapered amplifier for cold atom experiments**Project supported by the National Natural Science Foundation of China (Grant No. 61473166).

Simultaneous two-frequency amplification is highly desirable in cold atom experiments. The nonlinear response would appear in the two-frequency amplification with a semiconductor tapered amplifier (TA) and has a direct influence on the experimental result. We investigated in detail the effects of frequency difference, total power, and power ratio of two seeding lasers on the output components based on a simplified theoretical model. The simulation results showed that the multiple sideband generation in the amplifier due to self-phase and amplitude modulation could be suppressed and the TA tended to linearly amplify the power ratio between two-frequency components, when the two seeding lasers had a large frequency difference. This was verified experimentally in the output power ratio measurement via a calibrated Fabry–Perot interferometer method with a good linearity and an uncertainty of 1%. We also discussed the consequences of power ratio responses in the amplification in light of cold atom experiments, especially in the ac Stark shift related phase error of Raman-type atom interferometers (AIs). It was shown that the fluctuation of intensity ratio of Raman beams may induce significant systematic errors for an AI gyroscope.

Modal gain equalization of 18 modes using a single-trench ring-core EDFA

The few-mode erbium-doped fiber amplifier (FM-EDFA) is a key component of a few-mode-fiber-based optical communication system to allow the simultaneous amplification of all signal mode groups. This paper proposes the single-trench ring-core FM-EDFA for gain equalization of five signal mode groups—LP01, LP11, LP21, LP31, and LP41. The proposed fiber with ring-core Er+3 doping and fundamental pumping is used to study the gains and differential modal gains (DMGs) of signal mode groups. In this study, we have shown that introduction of a trench increases the modal confinement of higher mode groups and decreases that of lower mode groups in the ring region (or doped region) slightly. This helps in controlling the DMG and also increases the effective index difference Δneff between the adjacent modes. We show that at 1530 nm signal wavelength, the DMG of five mode groups is reduced from 1.32 dB (without trench) to 0.45 dB (with trench) with gain in excess of 20 dB. Also, the mode spacing Δneff increases from 4.9×10−4 to 5.1×10−4. Over the C-band, we have achieved more than 20 dB amplification and nearly 1 dB gain excursion while maintaining sufficient mode spacing Δneff>5.1×10−4 to avoid mode coupling due to macrobending.

JAK2 double minutes with resultant simultaneous amplification of JAK2 and CD274 in a therapy‐related myelodysplastic syndrome evolving into an acute myeloid leukaemia

<i><scp>JAK</scp>2</i> double minutes with resultant simultaneous amplification of <i><scp>JAK</scp>2</i> and <i><scp>CD</scp>274</i> in a therapy‐related myelodysplastic syndrome evolving into an acute myeloid leukaemia

Metagenome complexity and template length are the main causes of bias in PCR-based bacteria community analysis.

Multitemplate PCR is used widely for the study of microbial community diversity. Although such studies have established the abundance of different groups within many natural ecosystems, these reports are limited by uncertainties such as bias and artifacts in the PCR. Bias which is introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. In this study, factors leading to the bias of the multitemplate PCR in bacterial communities were examined and optimized. Comparisons between PCR cycle parameters, DNA polymerases, PCR primer degeneracy, and 16S rRNA gene fragments GC content, revealed that annealing temperatures and DNA structure are predominant factors contributing to the observed bias. Pre-digestion of metagenomic DNA with the restriction enzyme Sau3A I and decreased annealing temperature reduced the bias significantly. The application of these optimized conditions to the ten-species model community in a soil sample verified the validity of these treatments.

Optimization of high-power Er/Yb co-doped fiber amplifiers for dual-band pulse amplification

In this paper, high-power pulse-pumped erbium–ytterbium co-doped fiber amplifiers (EYDFAs) for simultaneous amplification of low-repetition rate pulses in the 1.0 [Formula: see text]m and 1.5 [Formula: see text]m bands are numerically investigated. The influences of the peak power and pulse-width of the pump and time delay between the pump and signal pulses on the performance of the amplifier are systematically analyzed. The results show that a pulse-pumped EYDFA is capable of simultaneously amplifying dual-band pulses. The optimal pulse-width of the pump is mainly determined by the allowable power of the backward Yb amplified spontaneous emission (ASE). By simply adjusting the time delay between the pump and signal pulses, the power ratio of the dual-band signals can be easily changed. Such kind of dual-band-pulsed EYDFAs have great potential applications in fields such as the generation of visible or mid-infrared light by nonlinear frequency conversion, counter stealth Lidar, etc.

Long-haul optical transmission link using low-noise phase-sensitive amplifiers

The capacity and reach of long-haul fiber optical communication systems is limited by in-line amplifier noise and fiber nonlinearities. Phase-sensitive amplifiers add 6 dB less noise than conventional phase-insensitive amplifiers, such as erbium-doped fiber amplifiers, and they can provide nonlinearity mitigation after each span. Realizing a long-haul transmission link with in-line phase-sensitive amplifiers providing simultaneous low-noise amplification and nonlinearity mitigation is challenging and to date no such transmission link has been demonstrated. Here, we demonstrate a multi-channel-compatible and modulation-format-independent long-haul transmission link with in-line phase-sensitive amplifiers. Compared to a link amplified by conventional erbium-doped fiber amplifiers, we demonstrate a reach improvement of 5.6 times at optimal launch powers with the phase-sensitively amplified link operating at a total accumulated nonlinear phase shift of 6.2 rad. The phase-sensitively amplified link transmits two data-carrying waves, thus occupying twice the bandwidth and propagating twice the total power compared to the phase-insensitively amplified link.

Phenotypic and Genotypic Characterization of Mycobacteria Isolates from Buruli Ulcer Suspected Patients Reveals the Involvement of Several Mycobacteria in Chronic Skin Lesions

is a cutaneous mycobacterial disease that occurs in tropical countries in sub-Saharan Africa, South-East Asia, Australia and America. The responsible pathogen is Mycobacterium ulcerans. Cote d'Ivoire is the most affected country, with more than 30 endemic health districts reporting a large number of chronic skin lesions. The clinical forms and the severity of ulcers vary from one patient to another. Samples from suspected patients were analyzed by PCR at Pasteur Institute of Cote d'Ivoire, as recommended by WHO. IS2404 sequence was detected in 61% of cases, incriminating M. ulcerans in chronic cutaneous lesions. For the other cases the etiology was not identified, thus raising several questions. Are all reported Buruli ulcer cases really caused by M. ulcerans? Would other mycobacteria be involved in the occurrence of chronic skin lesions considered as Buruli ulcer? BU suspected patients were enrolled in endemic areas of Cote d’Ivoire. Samples were collected from cutaneous lesions and transported to the lab at +4°C in 2 ml of Middlebrook 7H9 medium supplemented by Cetylpiridium chloride. The centrifugation pellet was taken with saline buffer to perform microscopic examination and mycobacteria isolation on Lowenstein-Jensen medium. Biochemical characteristics were described by the nicotinic acid detection according to Konno protocol, by the Nitrates reduction test, by the catalase activity detection at 22° and 68°C and the Wayne's Tween 80 hydrolysis test. Genotypic characteristics were determined by PCR with 1 ml of bacterial suspension targeting the insertion sequences (IS6110, IS2404, and IS2606), the plasmid virulence genes and Miru-VNTR loci (Miru-1, VNTR 6, VNTR 19, ST-1). A total of 47 mycobacterial strains were isolated with 3 different types of colonies whose microscopic examination showed Acid-Alcohol-Resistant Bacilli. 65.9% of isolates expressed biochemical characters in favor of M. ulcerans strains and 6.4% in favor of M. marinum strains. For 29.8% of isolates, the characteristics were related to atypical mycobacterial species. The genotyping targeting the IS6110, IS2606 and IS2404 insertion sequences allowed simultaneous amplification in 53.2% of isolates. IS2404 was amplified in 93.6% of isolates; IS6110 was amplified in 74.5% of isolates and IS2606 was amplified in 70.2% of isolates. Five genotypes were identified corresponding to various species of mycobacteria: genotypes 1 and 2 accounted for 63.8% with all the 3 insertion sequences and biochemical characteristics in favor of M. ulcerans strains; genotype 4 accounted for 6.4% of isolates with insertion sequences and biochemical characteristics in favor of M. marinum strains; The strains of genotypes 3 and 5 expressed molecular and biochemical characters relating to various non-M. ulcerans mycobacteria. Virulence genes were found in 72.3% of isolates corresponding to 90% of M. ulcerans strains and 60.7% of non-M. ulcerans mycobacteria. This study confirmed the involvement of several genotypes of M. ulcerans and other mycobacteria in chronic cutaneous lesions suspected as cases of in Cote d’Ivoire.

Comparative Analysis of Verification Parameters of Event-Specific Methods in GMO Maize

The second most dominant genetically modified (GM) crop is maize. Increasing number of GM maize events puts significant pressure on GMO testing laboratories to achieve the level of competence necessary to fulfill legal requirements. In the European Union, Polymerase Chain Reaction (PCR) is the method of choice for identification and quantification of GMOs. We performed verification of validated methods for identification of four GM maize events. Additionaly we aimed to explore the option of designing a method for simultaneous detection of these events in a multiplex PCR reaction. DNA was extracted from certified reference materials (CRM) using validated CTAB extraction protocol. Concentration of DNA was measured using Qubit dsDNA Broad Range Assay. Amplification of taxon specific marker for maize and all event-specific methods was performed according to the JRC Compendium of Reference Methods for GMO Analysis. Absolute limit of detection (LODabs) was determined for taxon specific and four event specific RealTime PCR based methods. DNA extracted from CRMs showed sufficient concentration for downstream analyses and preparation of dilutions for determination of LODabs. Determined LODabs for all tested methods meet acceptance criteria. As expected, the methods performance with respect to the repeatability and precision decline with the decrease in concentration of the target. Event-specific GA21 and NK603 methods show high Ct values at the determined LODabs. However, by adjusting the concentrations of primers and probes sensitivity of these two methods should be improved. Considering that the amplicons for all five methods are quite short (&lt;120 bp) optimization of multiplex reaction conditions for simultaneous amplification should be feasible.

Parallel Tests Using Culture, Xpert MTB/RIF, and SAT-TB in Sputum Plus Bronchial Alveolar Lavage Fluid Significantly Increase Diagnostic Performance of Smear-Negative Pulmonary Tuberculosis.

At present, tuberculosis remains a serious threat to human health. The diagnosis of pulmonary tuberculosis (PTB) is still difficult, and the prominent challenge for diagnosis is the lack of a highly sensitive and specific method. In order to explore the diagnostic value of parallel tests, this study prospectively enrolled 258 patients with smear-negative PTB from May 2, 2015 to December 31, 2016. The sputum specimens and bronchial alveolar lavage fluid (BALF) samples from all patients were assessed for MTB detection by culture, Xpert MTB/RIF, and simultaneous amplification and testing method for TB (SAT-TB). Overall, the sensitivity of any single test using culture, Xpert MTB/RIF, or SAT-TB was lower than that for parallel tests (p < 0.05), and the sensitivity rates for MTB detection in BALF were significantly higher than those in sputum samples. There were lower agreements in the detection results between sputum samples and BALF for all tests (p < 0.05). The parallel tests models of using culture plus Xpert MTB/RIF plus SAT-TB, culture plus Xpert, or culture plus SAT-TB achieved higher sensitivities compared with all three single test models (p < 0.05). Additionally, joint detection using sputum and BALF samples achieved a high sensitivity (0.8566, 95% CI: 0.8086–0.8941). In conclusion, the parallel tests model using culture, Xpert MTB/RIF, and SAT-TB in sputum plus BALF significantly increases the diagnostic performance of smear-negative PTB; thus, this method should be applied clinically when PTB is suspected but smear results are negative.

Gate-Controlled Graphene-Silicon Schottky Junction Photodetector.

Various photodetectors showing extremely high photoresponsivity have been frequently reported, but many of these photodetectors could not avoid the simultaneous amplification of dark current. A gate-controlled graphene-silicon Schottky junction photodetector that exhibits a high on/off photoswitching ratio (≈104 ), a very high photoresponsivity (≈70 A W-1 ), and a low dark current in the order of µA cm-2 in a wide wavelength range (395-850 nm) is demonstrated. The photoresponsivity is ≈100 times higher than that of existing commercial photodetectors, and 7000 times higher than that of graphene-field-effect transistor-based photodetectors, while the dark current is similar to or lower than that of commercial photodetectors. This result can be explained by a unique gain mechanism originating from the difference in carrier transport characteristics of silicon and graphene.

Conversion from non-orthogonally to orthogonally polarized optical single-sideband modulation using optically injected semiconductor lasers.

This Letter investigates an optically injected semiconductor laser for conversion from non-orthogonally to orthogonally polarized optical single-sideband modulation. The underlying mechanism relies solely on nonlinear laser characteristics and, thus, only a typical semiconductor laser is required as the key conversion unit. This conversion can be achieved for a broadly tunable frequency range up to at least 65GHz. After conversion, the microwave phase quality, including linewidth and phase noise, is mostly preserved, and simultaneous microwave amplification up to 23dB is feasible.

Clinical application of a protocol based on universal next-generation sequencing for the diagnosis of beta-thalassaemia and sickle cell anaemia in preimplantation embryos.

Mutations of the beta-globin gene (HBB) cause beta-thalassaemia and sickle cell anaemia. These are the most common cause of severe inherited disease in humans. Traditional preimplantation genetic testing protocols for detecting HBB mutations frequently involve labour intensive, patient-specific test designs owing to the wide diversity of disease-associated HBB mutations. We, therefore, asked the question whether a universally applicable preimplantation genetic testing method can be developed to test for HBB gene mutations. A multiplex polymerase chain reaction protocol was designed, allowing simultaneous amplification of multiple overlapping DNA fragments encompassing the entire HBB gene sequence in addition to 17 characterized, closely linked single nucleotide polymorphisms (SNP). Amplicons were then analysed using a next-generation sequencing method, revealing mutations and SNP genotypes. The protocol was extensively validated, optimized and eventually clinically applied on whole-genome amplified DNA derived from embryos of three couples carrying different combinations of beta-thalassaemia mutations. The HBB mutation status and associated SNP haplotypes were successfully determined in all 21 embryos. Analysis of 141 heterozygous sites showed no instances of allele dropout and the test displayed 100% concordance compared with the results obtained from karyomapping. This suggests that the combination of trophectoderm biopsy and highly sensitive next-generation sequencing may provide superior accuracy than typically achieved using traditional preimplantation genetic testing methods. Importantly, no patient-specific test design or optimization was needed. It is hoped that protocols that deliver almost universally applicable low-cost tests, without compromising diagnostic accuracy, will improve patient access to preimplantation genetic testing, especially in less affluent parts of the world.

Application of SAT technique in detection of Chlamydia trachomatis and Ureaplasma urealyticum

Objective To study the Chlamydia trachomatis (CT) and Ureaplasma urealyticum (UU) infection in Guangzhou area, and analyze the consistency of simultaneous amplification and testing (SAT) and conventional methods (CT was detected by latex immunochromatography, UU was detected by liquid culture method). Methods A total of 12 120 samples of urogenital secretions or urine samples were collected from Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University from January 2015 to December 2016. CT-RNA and UU-RNA were detected by the SAT technique, a part of samples were tested by conventional methods at the same time. The positive rates of CT and UU by SAT and the conventional methods between different gender and age groups were analyzed by χ2 test, the consistencies between different detection methods were analyzed by Kappa test. Results The positive rate of CT was 4.05%(356/8 781), UU 33.69%(1 125/3 339)in Guangzhou from 2015 to 2016. The positive rate of UU was significantly higher than that of CT (χ2=1 981, P<0.01). Of 145 specimens for CT test, the coincidence rate between SAT and latex immunochromatographic method was 96.55%(140/145), which showed good consistency (Kappa=0.65). Of 186 specimens for UU test, the coincidence rate of the results between the SAT method and liquid culture was 92.47%(172/186), which showed strong consistency (Kappa=0.81). Conclusions The positive rate of UU was significantly higher than that of CT in Guangzhou. The SAT method and conventional methods to detect CT and UU show high consistency, which can provide the evidence for clinical diagnosis of CT and UU infection.(Chin J Lab Med, 2018, 41: 380-384) Key words: Chlamydia trachomatis; Ureaplasma urealyticum; Bacteriological techniques; Nucleic acid amplification techniques; Immunochromatography

Exploring DNA quality of single cells for genome analysis with simultaneous whole-genome amplification

Single cell genome analysis methods are powerful tools to define features of single cells and to identify differences between them. Since the DNA amount of a single cell is very limited, cellular DNA usually needs to be amplified by whole-genome amplification before being subjected to further analysis. A single nucleus only contains two haploid genomes. Thus, any DNA damage that prevents amplification results in loss of damaged DNA sites and induces an amplification bias. Therefore, the assessment of single cell DNA quality is urgently required. As of today, there is no simple method to determine the quality of a single cell DNA in a manner that will still retain the entire cellular DNA for amplification and downstream analysis. Here, we describe a method for whole-genome amplification with simultaneous quality control of single cell DNA by using a competitive spike-in DNA template.

A film-based integrated chip for gene amplification and electrochemical detection of pathogens causing foodborne illnesses

Given the increased interest in public hygiene due to outbreaks of food poisoning, increased emphasis has been placed on developing novel monitoring systems for point-of-care testing (POCT) to evaluate pathogens causing foodborne illnesses. Here, we demonstrate a pathogen evaluation system utilizing simple film-based microfluidics, featuring simultaneous gene amplification, solution mixing, and electrochemical detection. To minimize and integrate the various functionalities into a single chip, patterned polyimide and polyester films were mainly used on a polycarbonate housing chip, allowing simple fabrication and alignment, in contrast to conventional polymerase chain reaction, which requires a complex biosensing system at a bench-top scale. The individual integrated sensing chip could be manually fabricated in 10 min. Using the developed film-based integrated biosensing chip, the genes from the pathogens causing foodborne illnesses were simultaneously amplified based on multiple designed microfluidic chambers and Hoechst 33258, which intercalates into double-stranded DNA, to generate the electrochemical signal. The target pathogen gene was accurately analyzed by square wave voltammetry (SWV) within the 25 s, while the gel electrophoresis required about 30 min. Based on the developed integrated biosensing chip, the 1.0 × 101 and 1.0 × 102 colony-forming unit (CFU) of Staphylococcus aureus and Escherichia coli were sensitively detected with high reproducibility in the 25 s. On the basis of the significant features of the film-based molecular analysis platform, we expect that the developed sensor could be applied to the screening of various pathogens as a POCT device.

A High‐throughput Targeted Bisulfite Sequencing‐based Analysis for Epigenetic Age Quantification and Monitoring

Aging represents the most important risk factor for many chronic diseases including cardiovascular diseases, diabetes, and cancer, therefore understanding the mechanisms of aging is a fundamental step for designing new treatments for chronic diseases. DNA methylation is the most reliable and accurate molecular marker for aging quantification, however, genome‐wide DNA methylation profiling techniques, such as reduced representative bisulfite sequencing and Illumina Bead Array that are widely used in aging research are prohibitively expensive and have poor data quality at low‐read coverage sites. Here we report a robust targeted bisulfite sequencing approach, called SWARM™ (Simplified Whole‐panel Amplification Reaction Method), for the accurate biological age determination. SWARM™ is flexible and low cost, requires relatively low DNA input, allows the simultaneous amplification and sequencing of hundreds of loci, and has shown to increase sample throughput. Using the SWARM™ approach, we were able to analyze the methylation level of several hundreds of age‐associated loci including the published Horvath Clock sites. An age‐predictive model was built using the elastic net regression of DNA methylation levels of the loci and chronological age of blood DNA samples of over 300 healthy subjects of 18 to 89 years old. Our epigenetic age (DNAge™) predictor achieved a small median age error, and the DNAge™ of samples that were processed on both sequencing and array platforms highly correlated (r &gt; 0.9). In brief, our versatile SWARM™ technology‐based DNAge™ platform is a very fast and accurate tool for the precise biological aging quantification and aging interventions monitoring. Dedicated DNAge™ platforms are compatible with various sample types, and can be applied for aging studies in human, and in other species including mice.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Multiplex polymerase chain reaction in combination with gel electrophoresis-inductively coupled plasma mass spectrometry: A powerful tool for the determination of gene copy number variations and gene expression changes

During the last few years multiplex real-time or quantitative polymerase chain reaction PCR (qPCR) has become the method of choice for multiplex gene expression changes and gene copy number variations (CNVs) analysis. However, such determinations require the use of different fluorescent labels for the different amplified sequences, which increases significantly the costs of the analysis and limits the applicability of the technique for simultaneous amplification of many targets of interest in a single reaction. In this regard, the use of the coupling between gel electrophoresis (GE) separation with inductively coupled plasma mass spectrometry (ICP-MS) detection allows the label-free determination of multiplex PCR-amplified sequences (amplicons) by monitoring the P present in the DNA backbone. The quantitative dimension is obtained since under optimal and controlled multiplex PCR conditions the peak areas of the separated amplicons are directly proportional to the amount of DNA template in the original sample. Moreover, the calibration of the GE-ICP-MS system with a DNA ladder permits direct estimation of the size (bp) of the PCR products. The suitability of the proposed multiplex strategy has been evaluated addressing two different situations: determination of CNVs and gene expression changes in human ovarian cancer cells. In the first case, the results obtained for the simultaneous quantitation of CNVs of four genes (HER2, CCNE1, GSTM1, ACTB) on DNA obtained from OVCAR-3 cells were in accordance with the literature data, and also with the results obtained by conventional simplex qPCR. In the second case, multiplex gene expression changes of BAX, ERCC1 and CTR1 genes, using ACTB as constitutive gene, on A2780cis respect to A2780 cells, resistant and sensitive to cisplatin, respectively, provided the same information as single reaction reverse transcription (RT)-qPCR.

Ultrahigh Frequency Nanomechanical Piezoresistive Amplifiers for Direct Channel-Selective Receiver Front-Ends.

Channel-selective filtering and amplification in ultrahigh frequency (UHF) receiver front-ends are crucial for realization of cognitive radio systems and the future of wireless communication. In the past decade, there have been significant advances in the performance of microscale electromechanical resonant devices. However, such devices have not yet been able to meet the requirements for direct channel selection at RF. They also occupy a relatively large area on the chip making implementation of large arrays to cover several frequency bands challenging. On the other hand, electromechanical piezoresistive resonant devices are active devices that have recently shown the possibility of simultaneous signal amplification and channel-select filtering at lower frequencies. It has been theoretically predicted that if scaled down into the nanoscale, they can operate in the UHF range with a very low power consumption. Here, for the first time nanomechanical piezoresistive amplifiers with active element dimensions as small as 50 nm × 200 nm are demonstrated. With a device area of less than 1.5 μm2 a piezoresistive amplifier operating at 730 MHz shows effective quality factor ( Q) of 89,000 for a 50Ω load and gains as high as 10 dB and Q of 330,000 for a 250Ω load while consuming 189 μW of power. On the basis of the measurement results, it is shown that for piezoresistor dimensions of 30 nm × 100 nm it is possible to get a similar performance at 2.4 GHz with device footprint of less than 0.2 μm2.

Detection and incidence of Bordetella holmesii in respiratory specimens from patients with pertussis-like symptoms in New South Wales, Australia

Bordetella pertussis, the aetiological agent of whooping cough is routinely diagnosed by polymerase chain reaction (PCR) directed at IS481, an insertion sequence targetalso found in Bordetella holmesii. Recent reports have suggested that B.holmesii infections can be misdiagnosed as pertussis, which can have a significant impact on public health surveillance. This study investigated the presence of B.holmesii in B.pertussis positive clinical samples, in order to determine the incidence of B.holmesii. Clinical cases of pertussis diagnosed by IS481-specific PCR between October 2008 and March 2016 in New South Wales were included. Bordetella holmesii was detected through the simultaneous amplification of IS481 and B.holmesii specific insertions sequence, hIS1001. A total of 46 of 802 patients were identified to be positive for B.holmesii rather than B.pertussis, suggesting an incidence rate of 6.5% in 2009, 16.8% in 2010, 7.6% during 2013 and 8.1% during 2015. Bordetella holmesii infections were diagnosed during and between pertussis epidemics, however cases of B.holmesii and B.pertussis co-infections were not found. The predominant age group of B.holmesii infection was 11-18 years old, which was significantly different to the mean age of B.pertussis infections (0-6 years, p= 0.023). These findings revealed that B.holmesii was co-circulating alongside the B.pertussis epidemic for seven years, hidden from view, as B.holmesii infections have been diagnosed as B.pertussis. Confirmatory testing of B.pertussis positive samples for the presence of B.holmesii, especially during pertussis epidemics, should improve the quality of laboratory diagnosis and laboratory surveillance for pertussis. The presence of B.holmesii in Australia highlights the importance of testing for this pathogen and ongoing molecular surveillance that can guide the control of whooping cough.

PowerPlex® fusion 6C system: internal validation study

ABSTRACTThis work is aimed at describing the proceedings and parameters used to validate PowerPlex® Fusion 6C System, the polymerase chain reaction (PCR) amplification kit by Promega, for posterior implementation in the laboratorial routine of the Forensic Genetic Service. The PowerPlex® Fusion 6C System allows multiplex PCR, through simultaneous amplification and posterior detection by fluorescence of 27 loci. Characterization of the kit was made according to the laboratory's internal validation procedure based on validation guidelines from Scientific Working Group on DNA Analysis Methods. Some parameters were evaluated, such as specificity, analytical thresholds, sensitivity, precision, mixture studies, DNA control samples, a proficiency test and changes in the PCR-based procedures: final reaction volume and cycle number, changes in the reaction mixture for direct amplification. This kit proved to be very robust and the results are in concordance with previous developmental validation by the manufacturer. In some parameters, the results were better than expected.