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МОЛЕКУЛЯРНАЯ ГЕНЕТИКА, МИКРОБИОЛОГИЯ И ВИРУСОЛОГИЯ №3, 2013
29. Thompson J. D., Gibson T. J., Plewniak F. et al. Nucl. Acids Res. 1997; 24: 4876-82.
30. Whitehouse C. A. Antiviral Res. 2004; 64 (3): 145-60.
Поступила 08.06.12
GENETIC VARIANTS OF THE CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS CIRCULATING IN ENDEMIC AREAS OF THE SOUTHERN TAJIKISTAN IN 2009
I. D. Petrova1, Y. V. Kononova', E. V. Chausov1, A. M. Shestopalov1, F. H. Tishkova2
1 State Research Center of Virology and Biotechnology "Vector", Koltsovo, Novosibirsk Region, Russia; 2 Tajik Research Institute of Preventive Medicine, Ministry of Health of the Republic of Tajikistan, Dushanbe, Tajikistan
506 Hyalomma anatolicum ticks were collected and assayed in two Crimean-Congo hemorrhagic fever (CCHF) endemic regions of Tajikistan. Antigen and RNA of CCHF virus were detected in 3.4% of tick pools from Rudaki district using ELISA and RT-PCR tests. As of Tursunzade district, viral antigen was identified in 9.0% of samples and viral RNA was identified in 8.1% of samples. The multiple alignment of the obtained nucleotide sequences of CCHF virus genome S-segment 287-nt region (996-1282) and multiple alignment of deduced amino acid sequences of the samples, carried out to compare with CCHF virus strains from the GenBank database, as well as phylogenetic analysis, enabled us to conclude that Asia 1 and Asia 2 genotypes of CCHF virus are circulating in Tajikistan. It is important to note that the genotype Asia 1 virus was detected for the first time in Tajikistan.
Key words: CCHF virus, tick infection rate, phylogenetic analysis, genotype
© КОЛЛЕКТИВ АВТОРОВ, 2013
УДК 616.98:578.828.6]-092:612.017.1]-078.33
Rezvan Bagheri1*, Bahareh Rabbani2, Nejat Mahdieh3, Hossein Khanahmad4, Mansour Abachi5,
Soheila Asgari6
pCR-ELisA: A DIAGNosTIC AssAY For IDENTIFYING IRANIAN HIV seropositives
Reproductive Biotechnology Research Center, Avicenna Research Institute, Academic Center for Education, Culture and Research
(ACECR), Tehran, Iran. 2Genetic Medical Group, Faculty of Medicine, Ghazvin University of Medical Sciences, Ghazvin, Iran; 3Medical Genetic Group, Faculty of Medicine, Ilam University of Medical Sciences, Ilam, Iran; 4Department of anatomical sciences and molecular biology, Isfahan University of Medical Sciences, Isfahan, Iran; 5Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran; 6International campus, Tehran University of Medical Sciences, Tehran, Iran
Quantitative viral load monitoring is an important indicator of prognosis in human immunodeficiency virus type 1 (HIV-1). PCR-ELISA as a quantitative method is proven to be sensitive and specific for quantification of HIV-1. Extracted DNA of thirty seropositive and twenty seronegative individuals which were confirmed by ELISA and western blot were amplified with digoxigenin- labeled nucleotides; and then in ELISA procedure biotin-labeled probes were hybridized to the PCR products. Diluted PCR products were analysed by electrophoresis and ELISA methods. The observation revealed that combination of nested-PCR and ELISA leads to a sensitive and specific identification of three copies in HIV-infected; the specificity and sensitivity were 95% and 96.7%, respectively. In conclusion, PCR-ELISA was 10 fold more sensitive than nested-PCR. This study developed a high sensitive PCR-ELISA to assess the quantification of proviral DNA load in the most relevant case of HIV-1 subtype. The reproducibility and reliability of this high-throughput test makes it appropriate for general laboratories to use for quantification of viral load and clinical diagnosis.
Key words: HIV, PCR-ELISA, PCR, High Sensitivity
AIDS was recognized as a new emerging disease in the beginning of the 1980s and identified as a transmissible pathogen leading to death worldwide. Although global effort is used to control the AIDS pandemic, about 34 million people in the world are affected with human immunodeficiency virus (HIV) and approximately 2.7 million people are infected each year [1]. Determining the true prevalence of HIV-1 infection is a critical step in controlling and treatment of the disease; therefore there
is always a need to improve diagnostic methods of viral detection.
Many diagnostic procedures used to identify HIV infected individuals by testing blood products for antibodies against HIV antigen such as enzyme-linked immunosorbent assay (ELISA) [2] and western blot [3] techniques. The detection of human immunodeficiency virus type 1 (HIV) which relies on hybridization assays are not sensitive enough for diagnostic purposes. These resource-limited settings highly predict infection and are commonly applicable for diagnosis of HIV infection, however they have some weaknesses like representing false-negative results in acute HIV infection and false-positive in intact vaccine recipients and infants with HIV-seropositive mothers [4, 5]. Viral detection assays like antigen cultures and nucleic acid amplification procedures could also be used. PCR detection is increasingly used for the non-culture based laboratory diagnosis [6]. Neither antigen detection nor hybridization assays have reached a reasonable satisfactory point of sensitivity in term of viral detection [7]. To overcome the poor sensitivity and specificity of assays, an in vitro amplification method -nested PCR- would amplify the conserved sequences of HIV-1 proviral DNA providing high-quality sequences to identify the exact PCR fragments [8].
Quantitative methods have been developed to determine absolute quantification of RNA viral load [9] or pro-
*Correspondence Author:
Rezvan Bagheri (MSc), Avicenna Research Institute, P.O. Box: 19615-1177, Tehran, Iran. Postal code: 1936773493. E-mail: R.Bagheri@avicenna.ac.ir
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
viral DNA sequences in peripheral blood mononuclear cells (PBMCs) [10]. These assessments could be considered as an indicator during the follow-up of infected individuals; however, some techniques provide a rapid, sensitive and specific test which could be used for routine diagnostic analysis. Real-time PCR have already been developed to detect and quantify HIV nucleic acids in a wide variety of appliances, such as in cell culture, donated blood and serum samples [11, 12]. PCR-ELISA is set to offer an alternative method of detecting the viral load including the amplification of HIV DNA, hybridization in liquid medium with a specific probe and finally detection of hybridized product by ELlSA. In this study, a highly sensitive, PCR-ELISA assay was performed to quantitate HIV-1 specific PCR products. This methodology aimed to demonstrate if it could become easily practicable for routine clinical testing.
Material and methods
Sampling and DNA preparation
Thirty HIV-1 seropositive samples were confirmed by CD4+ cell count and ELISA. Twenty HIV-1 seronegative subjects were also taken at random from a sample of healthy individuals at low risk of HIV infection. A 5 ml sample of blood was taken from each individual into EDTA (0.5 M, PH=8); PBMC was collected from fresh blood by ficoll-hypaque gradient centrifugation. Cells were washed three times in phosphate-buffered saline (10 mM phosphate buffer, pH 7.2; 150 mM NaCl). The cell pellet was re-suspended in a solution of 20 mM Tris (pH 8.3). Cell counts were determined in a counting chamber, and samples were kept at -70°C for further processes. Finally DNA was extracted and purified from each PBMC pellet by High Pure Template PCR Kit (Roche, Mannheim, Germany).
DNA amplification
Nested-PCR was performed on target DNA of HIV with highly conserved sequences. The gag region-specific primer pairs were designed by Gene Runner (version 3.5, US) and BioEdit Software package (available through http://www.mbio.ncsu.edu/BioEdit/bioedit. html). The sequences of pair primers were as follows: first-round: JA152/155, JA152-F (5'-ATC TCT AGC AGT GGC GCC CGA ACA G-3'); JA155-R (5'-CTG ATA ATG CTG AAA ACA TGG GTA T-3') and second-round JA153/154, JA153-F' (5'-CTC TCG ACG CAG GAC TCG GCT TGC T-3'); JA154-R' (5'-CCC ATG CAT TCAAAG TTC TAG GTG A-3'). The first-round of PCR was performed in 25^l volume containing 1X PCR Buffer (10 Mm Tris-HCl, pH = 8.3, 50mM KCl), 1.5 mM MgCl2 (Invitrogen, Karlsruhe, Germany), 0.5 ^M of each primers JA152/JA155, 0.2 mM of each dNTPs (Roche, Mannheim, Germany), 2.0U Taq polymerase (Supper Taq, England), and 5 ^l of the template DNA. Initial denaturizing condition of this round was done at 95°C for 4 min, 30 cycles including 40 s at 94°C, 40 s at 55°C, 40 s at 72°C and a final extension at 72°C for 5 min. The mixture of the second-round contained the same components as the first-round and DIG labeling Mix including dNTPs and digoxigenin-11-dUTPs (Roche, Mannheim, Germany). Two microliter of the first round PCR product was used as a template DNA for the second-round amplification by primers of JA153/154. The second-round amplification was constructed from 4 min at 95°C for the initial denaturation, 25 cycles of 40 s 94°C, 40 s at 57°C, 40 s at 72°C and a final extension at 72°C for 5 min.
Detection of amplified products by ELISA
The detected products on 1.5% agarose gel were quantified by a rapid colorimetric hybridization of the HIV-1 DNA samples. Digox-igenin-labeled PCR products were identified using the commercially available PCR ELISA Dig Labeling Plus Kit (Roche, Mannheim, Germany). The method was optimized with 5'-end labeled-biotin probe P17: 5'-GAC TAG GGG AGG CTA GAA GGA GAG AGA TGG GTG CGA GA-3' which specified the 572 bp PCR products. Briefly, 20 |il of denatured solution was added to each reaction tube containing 3 ^L of digoxigenin-labeled PCR products and incubated for 10 min at 25°C. The denatured PCR products were mixed with hybridization
solution of 7.5 pmol/mL capture probe in a microcentrifuge tube. 200 ^l of the mixture was then added to streptavidin-coated microplate well and incubated for 1 hour at 50°C. The wells were washed 3 to 5 times with the washing solution. Two hundred microliters of anti-digoxigenin-POD antibody was added to each well and then plate was incubated at 37°C for 30 min. After the second wash, 200 ^l of TMB as a chromogenic substrate was added to each well and the plate was incubated for next 30 min at 37°C in the dark until a color change developed. The plates were evaluated using an ELISA reader (Anthos 2020, version 1.1, US), with the net absorbance of 450/620 nm.
Standardization
For quantifying the experiments, ten-fold serial dilutions ranging from 30x105 to 3 copies of the PCR products inserted in the cloning vector (InsT/Aclone™ PCR Product Cloning Kit), were prepared and subjected to the PCR-ELISA (Figure 1A). The cloned insert was amplified by nested PCR as fallows; JA151/156 primers, JA151-F (5'-ACT CTG GTA ACT AGA GAT CCC TCA GAC CC-3'); JA156-R (5'-TTC CTG AAG GGT ACT AGT AGT TCC TGC TAT-3') with the mentioned inner primers JA152/155. Serial dilution PCR products were qualified by conventional gel electrophoresis and microplate hybridization method. Sensitivity was assessed by standard curve based on known copy numbers (log base 10 scale) and then copy numbers of each individual was deducted from serial dilution curve.
Statistical analysis
Data were processed using SPSS ver.16 (SPSS, Chicago, IL, USA) and Graph Pad Prism 5.04 software (San Diego, CA, USA). The optimal cut off point was determined by Receiver Operating Characteristic (ROC) curves. /2 test was also used to evaluate the association between qualitative variables. Relationship between quantitative variables was assessed by Pearson coefficient.
Results and discussion
Specificity of PCR-ELISA
50 ng of purified DNA of all controls and patients were used to perform PCR-ELISA as this quantity of DNA avoided false negative results. Nested-PCR products of 572 bp were visualized by gel electrophoresis and ethid-ium bromide staining (data not shown). 50 ng of nested-PCR products was confirmed by ELISA to increase the sensitivity and the optical density of samples were measured (Table 1). The mean absorbance values of control group and positive group were 0.1011 and 1.5076 with SD ± 0.0214 and ±0.8846, respectively. In a patient case the highest value was nearly 3.76 OD (after the OD value of the substrate blank was subtracted for each microwell) (Table 1) while OD value below 0.144 (as discussed in the next section) could be related to negative control with great specificity.
Determination of PCR-ELISA positive/negative cutoff value
In accordance to the contamination-free guidelines, to prevent false-positive result all experiments were performed in triplicate and the data expressed mean values. The sensitivity and specificity values of PCR-ELISA test, based on the ROC curve (area under the curve: 0.97, 95% CI0.90-1.03), were 96.7% and 95%, respectively; while the cutoff point was determined as 0.144 OD. The results were consistent with the cut-off value which was calculated by the mean absorbance value of the negative controls (20 healthy individuals) plus three standard deviations [13]. The cut-off point represents a significant statistical difference between positive and negative cases (c2 = 42.01, p < 0.001) (Table 2).
Limit of detection in PCR and PCR-ELISA
To detect the limit of the PCR-ELISA, specimens were tested in parallel by ten-fold serial dilutions from 30 x 105 to 3 copies of cloned inserted DNA. The correlation
МОЛЕКУЛЯРНАЯ ГЕНЕТИКА, МИКРОБИОЛОГИЯ И ВИРУСОЛОГИЯ №3, 2013
2,5
Dilution 30x105 30x104 30x103 30x102 300 30 3 NC
OD 2,350 1,950 1,265 0,527 0,333 0,255 0,13 0,051
I 1'5
I 1
Я 0,5
Sample number Optical density Copy Numbers Calculated Sample number Optical density Copy Numbers Calculated
S1 3.567 >3 000 000 S16 1.063 45 339
S2 3.083 >3 000 000 S17 0.998 38 958
S3 1.063 45 339 S18 0.067 <3
S4 1.448 64 651 S19 0.992 38 332
S5 3.759 >3 000 000 S20 0.845 22 476
S6 2.891 >3 000 000 S21 0.875 25 654
S7 1.372 62 431 S22 1.182 54 466
S8 1.21 56 084 S23 1.416 63 702
S9 1.299 60 070 S24 0.805 18 453
S10 1.671 77 474 S25 1.438 64 348
S11 0.978 36 854 S26 1.557 68 905
S12 0.922 30 776 S27 0.744 13 000
S13 1.355 61 924 S28 2.664 >3 000 000
S14 0.827 20 629 S29 2.594 >3 000 000
S15 1.139 51 591 S30 1.406 63 414
HIV DNA Copies (Iog10)
Dilution 30x105 30x104 30x103 30x102 300 30 3 NC
OD
2,350 1,950 1,265 0,527 0,333 0,255 0,13 0,051
572 bp
Figure. Sensitivity of PCR-ELISA system for the quantitative detection of HIV-1 genome.
A) Pearson's correlation coefficient between the optical densities of the colorimetric reactions is plotted against HIV genome copy numbers (log 10).
B) Sensitivity of PCR fallowed by 1.5% agarose gel for 10-fold serial dilutions ranging from 30 x 105 to 3 (respectively lane 1 to 8) copies of the plasmid DNA containing HIV genes. There is no band in lane 8. Lane 9: 100
bp ladder, lane 10: positive control.
between result of PCR-ELISA (OD) and the log number of HIV DNA was significant (r2 = 0.98), with Pearson correlation coefficients of 0.95 (P < 0.0001) (Figure, A). The limit detection of PCR-ELISA was 3 copies of genomic DNA; whereas agarose gel electrophoresis did not have sufficient sensitivity for very low viral load (Figure, B) and the absorbance value for the sample with 3 copies of DNA could be misinterpreted as negative control in agarose gel. A rise in the sensitivity of PCR-ELISA in comparison with nested PCR revealed that PCR-ELISA was at least 10 times more sensitive than gel electrophoresis.
HIV infection cannot be detected in a low copy numbers and may be transmited to uninfected individuals. According to the evidences one question is whether high-quality and effective measurement of the viral load monitoring is needed. The present study aimed to manage antiviral treatment and diagnose HIV infection in at-risk patient. In the context of breast-fed infant, these diagnostic programs could be included in postnatal follow-up visit [14]. Also viral load in HIV vaccine recipients [15] and drug resistance cases could be targeted thorough treatment strategy [16]. PCR-ELISA procedure is designed by combination of two methods to amplify low quantities of DNA. Despite the exponential increase in diagnostic programs and due to the limitations in technical resources, a trend was made to move to real-
The results of optical density and calculated copy different individuals by pCR-ELisA
Table 1 numbers of
Sample number
Optical density
Copy Sample
Numbers f
number
Calculated
Optical density
Copy Numbers Calculated
51
52
53
54
55
56
57
58
59
510
511
512
513
514
515
3.567 3.083 1.063 1.448 3.759 2.891 1.372 1.21 1.299 1.671 0.978 0.922 1.355 0.827 1.139
>3 000 000 >3 000 000 45 339 64 651 >3 000 000 >3 000 000 62 431 56 084
60 070 77 474 36 854 30 776
61 924 20 629 51 591
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
1.063 0.998 0.067 0.992 0.845 0.875 1.182 1.416 0.805 1.438 1.557 0.744 2.664 2.594 1.406
45 339 38 958
<3 38 332 22 476 25 654 54 466
63 702 18 453
64 348 68 905 13 000
>3 000 000 >3 000 000 63 414
Range of detection: 3-3 000 000 copies of HIV.
time PCR. It is fast and approximately semi-automated; however, it is not sensitive and specific enough for this purpose [17]. Furthermore, it requires a high capital cost of equipment and expensive fluorescent probes which are more costly than a colorimetric PCR-ELISA format [18]. PCR-ELISA is valuable in laboratories which cannot afford real-time PCR technology. Comparison of the result of two different PCR methods depicted that real-time PCR with a substantial concordance although associated with a wide confidence interval, is slightly less sensitive than PCR-ELISA [19]. ELISA or real-time assay are the modified conventional PCR which represent a further development of the method, supporting highly specific results in a short time by using probes that increase the specificity of the assay. These approaches have a few limitations, such as high costs of equipment and relative sophistication analysis or lack of validated accuracy quality-control [20].
In this study we have highlighted a PCR-ELISA assay for detection of proviral HIV-1 DNA. This system, as a conventional technique, is not only powerful but also widely applicable as a quantitative method .The possibility of determining viral load in clinical specimens via the microplate hybridization assay could turn out
Table 2
Comparison of pCR-ELIsA and Western test
PCR-FTTSA
Negative (< 0,144) Positive (> 0,145)
Western
Total
negative positive
19 (95,0%) 1 (5,0%) 20 (100,0%)
1 (3,3%)
29 (96,7%)
30 (100,0%)
X2 = 42,01, p < 0,001
Sensitivity: 96,7%, specificity: 95,0%.
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
particularly useful in viral disease diagnosis, by providing a further diagnostic tool, when reliance upon serology alone for diagnosis of these diseases may be misleading or difficult to interpret. In vitro amplification methods generated by combination of nested PCR and ELISA enable us to identify low-viral load of proviral HIV-1 DNA. The nested PCR assay with an external standard has been used to detect obvious infection in individuals, when sub-optimal amounts of mononuclear cell DNA are present. Since the major problem of nested PCR alone is a great risk of contamination if no internal control is used [21]. Like other studies, final amplification of nested PCR methods using agarose gel electrophoresis does not have sufficient sensitivity for very low viral loads and they could be considered as false negative results. The sensitivity of the nested PCR is prompted by10-fold via ELISA detection [22, 23].
At present some commercially available molecular assays are developed as a quantitative viral load measurement. These quantitative PCR test in particular COBAS AMPLICOR HIV-1 MONITOR test (version 1.5) (Roche Molecular Systems, Inc.) [24] and APTIMA HIV-1 qualitative RNA assay (Gen-Probe Incorporated) that are FDA-approved have some limitation. The COBAS HIV-1 test can only detect 50 HIV-1 genome copies/ml and cannot be considerable in prognosis [25, 26]. Recently the new COBAS Ampliprep/CobasTaqMan assay has been developed as an easily applicable method for high-throughput without user intervention [27]. On the other hand, evaluated APTIMA RNA qualitative assay is recognized as a semi-quantitative method for HIV patients who underwent antiretroviral therapy. In this essay in accordance to their opinion processing efficiency and turnaround time would be essential to be proved for validated in commercial use [28]. Although, quantifying methods for antiretroviral therapeutic monitoring of HIV are high throughput, cost-effectiveness consideration should not be neglected. These limitations could be attributed to financial aspects [29].
In conclusion, in the presented method even low-viral load could be detected confidently. The study's final goal was to accomplish reproducible results for performing clinical practice. Thus, the assay may become easily practicable for routine clinical testing or may be useful for research. The microplate hybridization described here appears to be specific, highly sensitive and rapid, as it requires only a day to provide reliable results; therefore, it might also be feasible in routine diagnostic laboratories, as the mentioned procedures are simple and familiar to most laboratory operators.
Acknowledgments
We thank Drs B. Behbahani and R. Yaghubi from Shiraz University of Medical Sciences for their help and
H. Heidari-Vala from Avicenna Research Institute for cordially help in manuscript editing.
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