Comparison of quantitative methods of DNA CMV investigation in clinical samples obtained from symptomatic and asymptomatic patients undergo renal transplantation

Cytomegalovirus infection is one of the main problem in immunocompromised patiens. Quantitative assessment of CMV load (viral load), rate of increase of load and determination of DNA level above which the likelihood of disease is high (viral load thresholdfor disease) have significant prognostic and therapeutic importance at transplant recipients. The aim of this work was the comparison of 3 quantitative molecular techniques and assessment the threshold for disease for each of them. The study was undertaken with 37 samples of serum and the whole from 17 renal transplant recipients. Part of samples (n=16) comes from symptomatic patients, and were taken in period of clinical symptoms demonstration. The samples ware investigated by hybridization method (HC) performed accordingly to Hybrid the Capture procedure, (r-t PCR) Amplicor test (COBAS AMPLICOR CMV the Monitor test) nd real time PCR (r-t PCR). In 21 out of 37 samples DNA CMV was detected by all 3 methods, 2 samples gave concordant negative results. The CMV DNA level measured by all 3 methods was significantly higher (p < 0.05; t-Student test) in samples from symptomatic patients than from asymptomatic: 4.79 versus 3.58 for HC; 3.06 versus 1.36 for PCR-Amplicor and 4.23 versus 2.88 log DNA copies/ml for r-t PCR. The threshold for disease connected with high likelihood of disease (p < 0.05; Fisher test) was established at 4 log for r-t PCR method, 4,61 for hybridization and 3 log DNA CMV copies/ml for PCR Amplicor.     

Cobas Ampliprep/Cobas TaqMan HIV-1 v2.0 Assay: Consequences at the Cohort Level

Background

High-sensitive real-time PCR assays are routinely used to monitor HIV-1 infected subjects. Inter-assay discrepancies have been described at the low viral load (VL) end, where clinical decisions regarding possible virological rebound are based.

Methods

A retrospective study was performed to analyze frequencies of viral blips after transition to the COBAS Ampliprep/COBAS TaqMan v2.0 HIV-1 assay (Taqman v2.0) in patients with prior undetectable VLs as measured with the Roche Cobas Ampliprep Amplicor HIV-1 Monitor Test, v1.5 (Amplicor) and was evaluated in comparison to a group of patients monitored with the Abbott Real-time HIV-1 assay (Abbott RT) during the same period of time.

Results

85 of 373 patients with VLs below the limit of quantification with Amplicor had VLs >50 copies/mL after transition to the TaqMan v2.0 assay. Among these 74.1% had VLs ranging from 50–499 copies/mL, 22.9% had VLs >500 copies/mL. From 22 patients with initial Taqman v2.0 based VLs exceeding 500 copies/mL, 6 patients had VLs <20 copies/mL after novel VL measurement on a next visit. In our control group with VL quantification using the Abbott RT assay, only 1 patient became detectable and showed a VL of <40 copies/mL after new measurement.

Conclusions

Transition to the Taqman v2.0 assay was accompanied by an increase of quantifiable HIV-1 VLs in patients with long term viral suppression under antiretroviral therapy that might be attributed to technical shortcomings of the Taqman v2.0 assay. A high test variability at the low VL end but also beyond was observed, making meaningful clinical interpretation of viral blips derived from different assays difficult.     

Advantage of PCR for detecting low amounts of HBV DNA in patients' sera

Serum hepatitis B virus DNA (HBV DNA) is now the most important and reliable marker for monitoring viral replication. Quantitative detection of HBV DNA in serum is based on a commercial standardized solution hybridization assay (Genostics). In this work, we studied the sensitivity and specificity of this method, in comparison with the polymerase chain reaction (PCR) technique, for low-value HBV DNA serum samples. Fifty-four patients with or without HBV serological markers were divided into 4 groups according to their HBV DNA values.Genomic amplication was found to affect 2 conserved regions of the viral genome, the S and C regions. Samples with an HBV DNA concentration equal to or greater than 1.5 pg/ml were considered positive in the “Genostics” test. A total of 38% of patients considered negative in the quantitative assay (< 1.5 pg/ml) were found to be positive for HBV DNA in serum after PCR. Only 26% of patients with an HBV DNA concentration of between 1.5 and 10 pg/ml in the Genostics test had PCR-detectable viral DNA in serum. Some 56% of patients with HBV DNA values between 10 and 20 pg/ml were found to be positive after amplification. All patients whose HBV DNA values were above 20 pg/ml had PCR-detectable viral DNA in serum.Our PCR results suggest that the positive limit level of the Genostics test has to be re-evaluated. Indeed, for low values of HBV DNA (under 20 pg/ml and especially under 10 pg/ml), it is not possible to conclude about the positivity from the quantitative assay, and results have to be estimated according to the clinical and serological status of the patients. Moreover, PCR can be falsely negative because of methodological problems.Nevertheless, this study confirms that PCR does enable detection of the viral genome in HBV-seronegative patients and in “old” and “cured” HBV-infection marker carriers.     

实时定量RT-PCR对乙型肝炎病毒全长RNA(fRNA)的定量检测

目的:建立一种简便的定量检测慢性乙型肝炎患者血清中终止于聚腺苷酸化位点的乙型肝炎病毒全长RNA(f RNA)的方法。方法:选取53例未治疗的乙型肝炎患者及22例HBs Ag阴性的健康者为研究对象,使用锚定oligo-d T的引物对其血清中f RNA进行实时定量反转录PCR检测,统计分析其与HBV DNA、HBcr Ag和HBe Ag的相关性。结果:对f RNA进行实时荧光定量RT-PCR检测的下线为2.3 log copies/ml,标准曲线的相关系数为0.99(P0.0001)。53例乙型肝炎患者中,29例(54.7%)可以检测到f RNA,22例正常对照中没有检测到f RNA。27例HBe Ag阳性和/或高水平HBV DNA的患者全部检测到f RNA,26例HBe Ag阴性并且低水平HBV DNA的乙型肝炎患者中有2例(7.7%)检测到f RNA(P0.0001)。HBe Ag阳性患者血清中f RNA水平高于HBe Ag阴性患者(5.0±0.3 vs.2.9±0.4 log copies/ml,P0.001)。f RNA与HBV DNA/HBcr Ag具有显著相关性(r=0.905、0.881,P0.0001)。Hayashi's定量分析法I显示f RNA与HBV DNA相关性强于其与HBcr Ag的相关性。结论:与HBV DNA和HBe Ag一样,f RNA可作为常规检测判断HBV的复制水平并指导用药。     

Triplex real‐time polymerase chain reaction assay for detection and quantification of norovirus (GI and GII) and sapovirus

To improve detection of norovirus (NoVGI, NoVGII) and sapovirus (SaV), a simultaneous quantitative RT‐PCR method was established. This triplex real‐time PCR method was evaluated using a combination of optimized specific primers and probes. The performance of the developed PCR assay was equivalent to that of monoplex real‐time PCR across a broad dynamic range of 10²–10⁷ copies/assay using plasmid DNA standards. The limit of detection was 10² copies/assay. The quantitative value was comparable with that of monoplex real‐time PCR of stool samples. Our triplex real‐time PCR is useful for detection of NoV and SaV infections.     

Application of nested PCR and mass spectrometry for DNA-based virus detection: HBV-DNA detected in the majority of isolated anti-HBc positive sera

DNA preparations from three different groups of serum samples were examined for HBV-DNA via a nested polymerase chain reaction assay (lower detection limit: 10 viral genomes in 100 μl serum): Group I consisted of 11 uninfected control sera, group II consisted of sera obtained from 11 HBV infected patients and group III consisted of 21 isolated anti-HBc positive samples. The 21 samples from group III were HBV-DNA negative according to a conventional non-nested PCR assay and hybridization with a ³²P-labelled probe. Using nested PCR and mass spectrometry, HBV-DNA was detected in none of group I and in all of group II samples. In 11 out of 21 (52%) of the isolated anti-HBc positive sera from group III, HBV-DNA was detected. No correlation was observed between HBV-DNA positivity and anti-HBc titers. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry provided a fast, sensitive and non-radioactive assay for the detection of PCR products without the need for gel electrophoresis or hybridization with labelled probes.     

Development and evaluation of a new detection tool—visual DNA microarray for simultaneous and specific detection of human immunodeficiency virus type-1 and hepatitis C virus

Human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health. Based on multiplex asymmetrical PCR and coupled with gold labelled silver stain (GLSS), we developed the visual DNA microarray for sensitive and specific detection of these two viruses. Capturing probes of 5′-end-amino-modified oligonucleotides were immobilized on glass surface to bind the complement biotinylated target DNA. The Au–streptavidin probe was introduced to the microarray for specific binding to biotin. Black images of microarray spots which result from the precipitation of silver onto Au–streptavidin probes, were visualized by naked eyes. In order to improve the efficiency of microarray hybridization, triplex asymmetrical PCR of HIV-1, HCV and Human enterovirus 71 (EV-71, used as positive control) were performed to prepare abundant biotinylated single-stranded target DNA. The sensitivity of visual DNA microarray (10³ copies/ml) was higher than conventional PCR (10⁴ copies/ml) and was identical to FQ-PCR (10³ copies/ml). Total 152 blood samples containing the two viruses were tested using the DNA microarray and fluorescence quantitative real-time PCR (FQ-PCR). The results were identical (P > 0.05). So this system has high sensitivity and may have potential in clinical applications.     

Rapid Real-Time PCR Assay for Detection and Quantitation of Mycobacterium avium subsp. paratuberculosis DNA in Artificially Contaminated Milk

Using fluorescence resonance energy transfer technology and Lightcycler analysis, we developed a real-time PCR assay with primers and probes designed by using IS900 which allowed rapid detection of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Initially, the PCR parameters (including primer and probe levels, assay volume, Mg²⁺ concentration, and annealing temperature) were optimized. Subsequently, the quantitative ability of the assay was tested and was found to be accurate over a broad linear range (3 × 10⁶ to 3 × 10¹ copies). The assay sensitivity when purified DNA was used was determined to be as low as five copies, with excellent reproducibility. A range of DNA isolation strategies was developed for isolating M. avium subsp. paratuberculosis DNA from spiked milk, the most effective of which involved the use of 50 mM Tris HCl, 10 mM EDTA, 2% Triton X-100, 4 M guanidinium isothiocyante, and 0.3 M sodium acetate combined with boiling, physical grinding, and nucleic acid spin columns. When this technique was used in conjunction with the real-time PCR assay, it was possible to consistently detect <100 organisms per ml of milk (equivalent to 2,000 organisms per 25 ml). Furthermore, the entire procedure (extraction and PCR) was performed in less than 3 h and was successfully adapted to quantify M. avium subsp. paratuberculosis in spiked milk from heavily and mildly contaminated samples.     

Evaluation of the NucliSens EasyQ v2.0 Assay in Comparison with the Roche Amplicor v1.5 and the Roche CAP/CTM HIV-1 Test v2.0 in Quantification of C-Clade HIV-1 in Plasma

Human immunodeficiency virus type 1 (HIV-1) genetic diversity poses a challenge to reliable viral load monitoring. Discrepancies between different testing platforms have been observed, especially for non-clade-B virus. Therefore we compare, in antiretroviral therapy (ART)-naïve South African subjects predominantly infected with HIV-1 clade-C, three commercially available assays: the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test version 2.0 by Roche (CAP/CTM v2.0), the BioMérieux NucliSens Version 2.0 Easy Q/Easy Mag (NucliSens v2.0) and the Roche COBAS Amplicor HIV-1 Monitor Test Version 1.5 (Amplicor v1.5). Strong linear correlation was observed and Bland-Altman analyses showed overall good agreement between the assays with mean viral load differences of 0.078 log cp/ml (NucliSens v2.0 – Amplicor v1.5), 0.260 log cp/ml (CAP/CTM v2.0 – Amplicor v1.5) and 0.164 log cp/ml (CAP/CTM v2.0 – NucliSens v2.0), indicating lower mean viral load results for the Amplicor v1.5 and higher mean readings for the CAP/CTM v2.0. Consistent with observations following previous comparisons of CAP/CTM v2.0 versus Amplicor v1.5, the CAP/CTM v2.0 assay detected low-level viremia (median 65 cp/ml) in more than one-third of those in whom viremia had been undetectable (<20 cp/ml) in assays using the NucliSens platform. These levels of viremia are of uncertain clinical significance but may be of importance in early detection of ART resistance in those on treatment. Overall the three assays showed good comparability of results but with consistent, albeit relatively small, discrepancies for HIV-1 clade-C samples, especially in the low-viremic range that should be taken into account when interpreting viral load data.     

Quantitation of Viral DNA by Real-Time PCR Applying Duplex Amplification, Internal Standardization, and Two-Color Fluorescence Detection

A real-time PCR method was developed to quantitate viral DNA that includes duplex amplification, internal standardization, and two-color fluorescence detection without the need to generate an external standardization curve. Applied to human parvovirus B19 DNA, the linear range was from 10² to at least 5 × 10⁶ copies per ml of sample. The coefficient of variation was 0.29 using a run control of 2,876 copies per ml. The method reduces the risk of false-negative results, yields high precision, and is applicable for other DNA targets.     

Performance of the automated COBAS AMPLICOR™ system for the detection of hepatitis C virus RNA

Background: The COBAS AMPLICOR^™ (CA) instrument for the amplification and detection steps of the AMPLICOR^™ molecular diagnostic assays has recently been introduced. The system contains a single thermal cycler with two independently controlled heating/cooling blocks, a pipettor, a magnetic particle washer, a photometer and an incubator.Objective: The performance of the CA instrument was evaluated in a routine diagnostic laboratory for the detection of hepatitis C virus (HCV) RNA. The new system was compared with the corresponding microwell plate assay (AMPLICOR HCV Test).Study design: Routine clinical sera (350) from hemodialysis patients and patients with chronic HCV infection and interferon therapy were studied. If discrepant results were obtained, both assays were repeated (specimen preparation, amplification and detection); in addition, the HCV copy number was determined with the AMPLICOR HCV MONITOR^™ Test.Results: There was a correlation between the CA HCV Test and the AMPLICOR HCV Test in 341 of 350 specimens (97%). After resolution of 9 discrepant results, the CA HCV Test gave a sensitivity of 97.8% and a specificity of 99.4%. The most common reason for discrepant results was a low HCV RNA copy number.Conclusion: The CA system was found to be a labor-saving, fast and reliable instrument for the amplification and detection steps of a RT-PCR molecular assay for detection of HCV RNA.     

A comparative study of PCR product detection and quantitation by electrochemiluminescence and fluorescence

Amplification and detection of target DNA sequences are made possible in a polymerase chain reaction (PCR) by using a mixture of biotinylated and ruthenium(II) trisbipyridal (Ru(bpy)₃²⁺)-end-labelled primers. In this way, biotin for capture and Ru(bpy)₃²⁺ for detection are directly incorporated into the PCR product obviating subsequent probe hybridization. PCR of a bacterial DNA template from Alteromonas species strain JD6.5 using a cocktail of biotin- and Ru(bpy)₃²⁺-labelled primers amplified a 1 kilobase region. Serial dilution of PCR product followed by magnetic separation with Streptavidin (SA)-coated magnetic beads and an electrochemiluminescence (ECL) assay using the semi-automated QPCR System 5000 demonstrated sensitive (pg range) DNA detection. ECL assay of probe hybridization to a human immunodeficiency virus (HIV) sequence also produced pg level sensitivity. Quantitative DNA determination by ECL assay correlated well with visual detection of DNA in electrophoretic gels. However, DNA detection by ECL assay was 10 to 100 times more sensitive than conventional ethidium bromide staining. The combination of DNA-based magnetic separation with ECL assay provides a very sensitive and rapid method of quantitating DNA which, owing to its rapid and facile nature, may have many applications in the research, environmental monitoring, industrial and clinical fields.     

Detection of Verotoxigenic Escherichia coli by Magnetic Capture-Hybridization PCR

Magnetic capture-hybridization PCR (MCH-PCR) was used for the detection of 36 verotoxigenic (verotoxin [VT]-producing) Escherichia coli (VTEC), 5 VTEC reference, and 13 non-VTEC control cultures. The detection system employs biotin-labeled probes to capture the DNA segments that contain specific regions of the genes for VT1 and VT2 by DNA-DNA hybridization. The hybrids formed were isolated by streptavidin-coated magnetic beads which were collected by a magnetic particle separator and, subsequently, amplified directly by conventional PCR. The detection system was found to be specific for VTEC: no amplification was obtained from non-VTEC controls, whereas VTEC isolates tested positive for one or two specific PCR products. With 5, 7, or 10 h of enrichment, the limits of detection were 10³, 10², and 10⁰ CFU/ml, respectively, by agarose gel electrophoresis. Southern hybridization did not seem to improve the limit of the detection. When applied to food, MCH-PCR was capable of detecting 10⁰ CFU of VTEC per g of ground beef with 15 h of nonselective enrichment. The results of MCH-PCR for pure cultures of VT1- and/or VT2-producing E. coli cells were in total agreement with toxin production as measured by a VT enzyme-linked immunosorbent assay.     

Abbott RealTime HBV Assay Is More Sensitive in Detection of Low Viral Load and Little Impacted by Drug Resistant Mutation in Chronic Hepatitis B Patients under Nucleot(s)ide Analogues Therapy

Background

Selection of drug-resistant strains may lead to failure of HBV antiviral therapy. There is little information whether there is detection difference in drug resistant mutations between different viral load assays of HBV.

Objectives

This study is aimed to investigate whether there is drug-resistant strains related detection difference between Abbott RealTime HBV (RealTime) and CobasAmpliPrep/CobasTaqMan HBV assays 2.0 (TaqMan).

Study Design

One hundred and thirty-four CHB patients who received HBV anti-viral therapy were enrolled. HBV virological markers were tested 3 months apart regularly. Serum HBV DNA levels were determined using the TaqMan and RealTime. YMDD (rt180M and rt204V) mutation was checked in patients who experienced virologic breakthrough (VBT).

Results

The correlation of HBV DNA observed between the RealTime and TaqMan was good for all 571 samples (R² = 0.797; P<0.001). However, the correlation in the 434 samples with HBV DNA level <3 log₁₀ IU/ml was not as good as in all samples (R² = 0.457). Overall, 21.5% of samples had a detection difference of ≥1 log₁₀ IU/ml with 91.9% of these having HBV DNA level <3 log₁₀ IU/ml. Twenty-four patients experiencedVBT. Three of these patients had acquired the YMDD mutation and exhibited discordant viral load results between the two methods tested. In each case, persistent HBV DNA was detected by RealTime and undetectable with TaqMan. Of the patients who experienced a VBT and had acquired YMDD mutation, 4.7% had undetectable HBV DNA by TaqMan while all were detectable with RealTime.

Conclusions

RealTime assay is more sensitive and is little impacted by the development of drug resistant mutation.     

Quantitation of herpes simplex virus DNA in cerebrospinal fluid of patients with herpes simplex encephalitis by the polymerase chain reaction

Background: Previous studies have shown the diagnostic utility of qualitative detection of herpes simplex virus (HSV) DNA by the polymerase chain reaction (PCR) in cerebrospinal fluid samples (CSF) from patients with herpes simplex encephalitis (HSE).Objectives: To determine whether quantitation of HSV DNA in CSF could be useful for monitoring efficacy of antiviral therapy and provide prognostic indications.Study design: A quantitative PCR assay using an internal control for evaluation of PCR efficiency and detection of PCR inhibitors was developed and used for retrospective testing of 98 CSF samples from 26 patients with serologically diagnosed HSE during the period 1980–1995.Results: HSV DNA was detected in 36 CSF samples from 23 patients. PCR positivity was 100% for CSF samples collected within 10 days after onset, and 30.4 and 18.7% for samples collected 11–20 and 21–40 days later, respectively. The 3 PCR-negative patients had their first CSF collected 14, 16, and 28 days after onset, respectively. Three of 98 (3.1%) CSF samples were completely or partially inhibitory to PCR. Initial DNA levels were not significantly different in patients with HSE due to either primary or recurrent HSV infection. In addition, they were not related to severity of clinical symptoms nor were predictive of the outcome. A progressive decrease in viral DNA levels was observed both in patients who received acyclovir therapy and in a small number of untreated patients.Conclusions: This study: (i) confirms the high sensitivity of PCR for the diagnosis of HSE; (ii) emphasizes the need for an internal control of amplification to achieve maximal sensitivity and perform reliable quantitation of viral DNA; and (iii) suggests that CSF might not be the best specimen to investigate in studies of the natural history of HSE.     

Quantitative detection of E. coli O157:H7 eaeA gene using quantum dots and magnetic particles

A quantitative gene detection technique targeting the pathogenic E. coli O157:H7 eaeA gene was developed using magnetic bead (MB)-quantum dots (QDs) nanoparticle complexes. MBs allowed for the separation of DNA-conjugated QD nanoparticles via magnetic field manipulation. QDs provided internal fluorescence calibration to account for the intrinsically different numbers of nanoparticles interrogated in each assay. Based on the measurement of normalized fluorescence (Cy3/QD₆₅₅), the linear quantification ranges of ssDNA and dsDNA targets were determined to be 10 through 10³ fM (R² = 0.992) and 2 × 10² through 6 × 10⁷ gene copies (R² = 0.972), with detection limits of 9.72 fM and 10⁴ gene copies, respectively. The kinetic results indicate that adjustment of hybridization temperature in accordance to the amount of target DNA was required to maximize the efficiency of DNA hybridization. We were able to discriminate perfectly matched target DNA, 1-, 2-, and 41-base pair mismatched target DNAs in our approach and therefore demonstrated excellent selectivity. Our technique was also used on pure bacterial culture to showcase its ability to analyze environmental samples.     

Technical and Regulatory Shortcomings of the TaqMan Version 1 HIV Viral Load Assay

Background

The lower limit of detection of the original Roche Amplicor HIV plasma viral load (pVL) assay (50 copies/mL) has defined HIV treatment success. The Amplicor assay, however, has been replaced by the Roche TaqMan assay(s). Changes to the limits of detection and calibration have not been validated for clinical utility. Sudden increases in the number of patients with detectable pVL have been reported following the introduction of the TaqMan version 1 assay.

Methods

Between October 2009 and April 2010 all routine pVL samples from British Columbia, Canada, with 40–250 copies/mL by TaqMan were re-tested by Amplicor (N = 1198). Subsequent short-term virological and resistance outcomes were followed in patients with unchanged therapy (N = 279; median 3.2 months follow-up).

Results

TaqMan and Amplicor values correlated poorly at low pVL values. Low-level pVL by TaqMan was not associated with impending short-term virological failure; only 17% of patients with 40–250 copies/mL by TaqMan had detectable pVL by Amplicor at follow-up. During the follow-up period only 20% of patients had an increase in pVL by TaqMan (median [IQR]: 80 [36–283] copies/mL). In addition, in ∼2.4% of samples pVL was dramatically underestimated by TaqMan due to poor binding of the proprietary TaqMan primers.

Conclusions

The replacement of Amplicor with the TaqMan assay has altered the previously accepted definition of HIV treatment failure without any evidence to support the clinical relevance of the new definition. Given the systematic differences in measurement in the low pVL range the British Columbia HIV treatment guidelines now use a threshold of >250 copies/mL by TaqMan to define treatment failure.     

Comparison of the Cobas Ampliprep/Cobas TaqMan HBV Test versus the Cobas Amplicor HBV monitor for HBV-DNA detection and quantification during antiviral therapy

Performances of the new automatic system COBAS AmpliPrep/COBAS TaqMan 48 (CAP/CTM) (Roche, Branchburg, NJ) for HBV DNA extraction and real-time PCR quantification were assessed and compared with the endpoint PCR COBAS AMPLICOR HBV Monitor (CAHBM, Roche). Analytical evaluation with proficiency panels from UK National External Quality Assessment Scheme (UK NEQAS) over a 1-year period of distribution showed that CAP/CTM correctly measured HBV DNA levels with a close correlation between expected and observed values (r=0.995). Clinical evaluation as tested with samples from 11 HBsAg-positive patients undergoing antiviral therapy (71 serial specimens of plasma), demonstrated excellent correlation with CAHBM (r=0.958, mean difference in quantitation: 0.14 log, IU/ml), but CAP/CTM detected longer period of residual viremia. HBV DNA reduction was much higher in the combination schedule (Lamivudine+Adefovir), than in Adefovir monotherapy (5.1 vs. 3.5 logs). In conclusion, CAP/CTM allows for an accurate and standardized quantification of HBV DNA in high through-put laboratories. Due to it high sensitivity, it may further improve the detection of emerging drug resistance strains and the assessment of antiviral therapy.     

Quantification and optimization of Candida albicans DNA in blood samples using Real- Time PCR

Background:

Candida albicans (C. albicans) is a major cause of candidaemia in people with impaired immunity. Blood culture is a “gold standard” for candidaemia detection but is time-consuming and relatively insensitive. We established a real-time PCR assay for C. albicans detection in blood by LightCycler PCR and melting curve analysis.

Methods:

Five milliliter blood samples from healthy volunteers were spiked with 10⁰-10⁶ C. albicans cells to determine the detection limit of our method. DNA was extracted from whole blood using glass beads and the QIAamp DNA Blood Mini Kit (Qiagen, Hilden Germany). DNA from C. albicans isolates were amplified with primers and inserted into Escherichia coli (E. coli) DH5α.1 cells with the TA cloning vector (Invitrogen). The plasmid was used for standardization and optimization. A quantitative PCR assay with the LightCycler amplification and detection system based on fluorescence resonance energy transfer (FRET) with two different specific probes was established. To assess the precision and reproducibility of real-time PCR the intra-assay precision was determined in six consecutive assays.

Results:

No cross-reactivity of the hybridization probes with the DNA of non-C. albicans species or human genomic DNA was observed, which confirmed its 100% specificity. The minimum limit detected was one C. albicans cell or 10⁰ CFU/ml (10 fg) per PCR reaction. The real-time PCR efficiency rate for Candida was high (E = 1.95). Melting curve analysis of C. albicans showed a specific melting peak temperature of 65.76 °C.

Conclusion:

The real-time PCR assay we developed is highly specific and sufficiently sensitive to detect the fungal load for early diagnosis of invasive candidiasis. Key Words: Invasive candidiasis, Real-time PCR, Candida albicans