KR100909621B1 - Real-Time PCR Analysis Method for Absolute Quantification of Human Bocavirus - Google Patents

Abstract

The present invention relates to a real time PCR method for the detection of human bocavirus (HBoV). Since the real-time PCR method according to the present invention can quickly identify NS1, NP-2 and VP1 genes of HBoV with high sensitivity, it can be usefully used for the diagnosis and prediction of infectious and pathogenic diseases of human respiratory diseases.

HBoV, real-time PCR

Description

Real-time PCR assays for absolute quantification for human bocavirus

1A and 1B are graphs showing standard curves and dynamic ranges for real-time PCR analysis.

2 shows the sensitivity of conventional PCR analysis for the HBoV NS1 gene.

Figure 3 shows the results of comparing the average C _T value in the clinical specimens positive in three assays.

1.Allander T, Tammi MT, Eriksson M, Bjerkner A, Tiveljung-Lindell A, Andersson B. 2005. Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc Natl Acad Sci U S A 102 (36): 12891-12896.

2. Arnold JC, Singh KK, Spector SA, Sawyer MH. 2006. Human bocavirus: prevalence and clinical spectrum at a children's hospital. Clin Infect Dis 43 (3): 283-288.

Bastien N, Brandt K, Dust K, Ward D, Li Y. 2006. Human Bocavirus infection, Canada. Emerg Infect Dis 12 (5): 848-850.

Choi EH, Lee HJ, Kim SJ, Eun BW, Kim NH, Lee JA, Lee JH, Song EK, Kim SH, Park JY, Sung JY. 2006.The association of newly identified respiratory viruses with lower respiratory tract infections in Korean children, 2000-2005. Clin Infect Dis 43 (5): 585-592.

5. Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA, Yao JD, Wengenack NL, Rosenblatt JE, Cockerill FR, 3rd, Smith TF. 2006. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev 19 (1): 165-256.

6.Kesebir D, Vazquez M, Weibel C, Shapiro ED, Ferguson D, Landry ML, Kahn JS. 2006. Human bocavirus infection in young children in the United States: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus. J Infect Dis 194 (9): 1276-1282.

Lu X, Chittaganpitch M, Olsen SJ, Mackay IM, Sloots TP, Fry AM, Erdman DD. 2006. Real-time PCR assays for detection of bocavirus in human specimens. J Clin Microbiol 44 (9): 3231-3235.

8. Ma X, Endo R, Ishiguro N, Ebihara T, Ishiko H, Ariga T, Kikuta H. 2006. Detection of human bocavirus in Japanese children with lower respiratory tract infections. J Clin Microbiol 44 (3): 1132-1134.

9. Sloots TP, McErlean P, Speicher DJ, Arden KE, Nissen MD, Mackay IM. 2006. Evidence of human coronavirus HKU1 and human bocavirus in Australian children. J Clin Virol 35 (1): 99-102.

10. Weissbrich B, Neske F, Schubert J, Tollmann F, Blath K, Blessing K, Kreth HW. 2006. Frequent detection of bocavirus DNA in German children with respiratory tract infections. BMC Infect Dis 6: 109.

11. Young NS, Brown KE. 2004. Parvovirus B19. N Engl J Med 350 (6): 586-597.

Field of technology

The present invention relates to a method for quickly and effectively detecting a virus associated with a human respiratory disease by a real-time PCR method.

Prior art

In 2005, a new human parvovirus, human bocavirus (HBoV), was discovered in Sweden using a randomized amplification method from pediatric patients with lower respiratory tract infections (LRTIs) [1]. HBoV is dug corruption against (parvoviridae) and parvovirus corruption for subfamilies, and Boca virus genus (bocavirus genus), the average genome size of 5 kbp of the envelope is a single stranded (non-enveloped single-stranded) the smallest with a DNA-free It is a kind of virus. Three open reading frames (ORFs) are located in the middle of the genome encoding nonstructural proteins (NS1, NP-1) and structural proteins (VP1 / VP2). [Supra: 1]. Parvovirus B19, Erythrovirus , is a hospital only for humans that causes fifth disease, arthrosis, transient aplastic crisis, persistent anemia, hydrops fetalis, and congenital anemia. It is an adult parvovirus (see supra: 11). HBoV is known as a human pathogenic parvovirus that is involved in respiratory diseases. Subsequent studies around the world have been reported on respiratory specimens obtained from children with respiratory tract infections [3, 4, 6, 8, 9 and 10]. This suggests that HBoV can become a pandemic virus worldwide. Therefore, a fast and accurate detection method is required for the diagnosis. However, exact infectious and clinical characteristics have not been identified to date. The only way to detect this virus is known as conventional PCR and real-time PCR, since HBoV culture and serological methods have not been established. Although the virus culture method is known to be a "gold standard" in laboratory diagnosis, this method has the disadvantage of being time consuming and having low sensitivity and specificity. In general, real-time PCR is advantageous in that it is fast in diagnosis, absolute quantitative, amplification and analysis in a closed system free of contamination, post-analysis is unnecessary, and many samples can be processed at once. There is this. [Supra: 5]. Previous studies have suggested a real-time PCR method with high sensitivity and specificity that targets NS1 and NP-1 genes [7]. In the present invention, the inventors of the present invention present a real-time PCR assay based on the Taq-Man method, which is highly sensitive and specific for the rapid detection of three ORFs of HBoV (NS1, NP-1 and VP1 genes) and the absolute absolute of virus in clinical respiratory specimens. To provide.

It is an object of the present invention to provide a real-time PCR method for the rapid detection of three ORFs (NS1, NP-1 and VP1 genes) of HBoV.

Another object of the present invention is to provide a method for quantifying HBoV with high sensitivity in clinical respiratory specimens.

It is another object of the present invention to provide a kit for detecting HBoV.

In order to achieve the above object, the present inventors have developed a specific quantitative real-time PCR assay for the detection and quantification of HBoV in patients with respiratory diseases. Three independent real-time PCR assays were designed to amplify the HBoV NS1, NP-2 and VP1 genes. For clinical evaluation, nasal aspirates from 102 children with winter 2006/2007 acute respiratory tract infections were tested in three quantitative real-time analyzes. Analyzes were performed with a wide dynamic range of 50 to 5 × 10 ⁷ copies each. The limit of detection was 10 genome copies and there was no cross reaction with other respiratory viruses and bacteria. In addition, high assay reproducibility was confirmed in inter / intraassay precision. Clinical evaluation identified 5 patients (4.9%) with approximately 10 ⁴ to 10 ⁸ copies of the virus identified in the analysis, and were identified as common cold, pneumonia, pharyngitis and sinusitis, respectively.

As such, the quantitative real-time PCR analysis of the present invention provides a sensitive and specific diagnostic means. The sensitivity of the three assays was 10 genomes increased 2-log units over the conventional PCR assay (FIG. 2). In each assay that performed linear amplification over a broad dynamic range with low intra / inter assay differences, no cross-reactivity with other DNA viruses and respiratory bacteria was observed. To determine the positive criteria, we repeated the clinical sample three times and considered positive as three independent assays to rule out possible false positives. However, in some samples VP1 gene target analysis showed high C _T values of less than 10 gene copies, which prevented the determination of positive results. We considered them as negative results. In addition, PCR premixes, control plasmid DNA and clinical sample processing were performed in separate locations to prevent possible contamination. Although conducted in a closed environment, the real-time PCR, for the other levels of the amplification products antifouling present inventors uracil in PCR Master Mix - N - glycoside sila imposed dehydratase (uracil- N -glycosylase) [supra: 5]. Clinical evaluation of the analyzes performed on 102 respiratory specimens obtained from patients with acute respiratory infection showed 5 human bocavirus positive results. These results were identical to those of conventional PCR, and the mean viral load of each sample in independent assays ranged from 3 x 10 ⁴ to 2 x 10 ⁸ copies per ml of sample (Table 3). None were positive. In this study, it was difficult to generalize due to the limited number of samples, but Lu et al. [7] reported that HBoV virus PCR positive patients with pneumonia have a relatively low viral load. Given all of the HBoV detected in patients with diverse spectrum of viral loads and infected respiratory tracts, there is a need for robust detection methods that are more sensitive, specific and have higher throughput processing than conventional PCR assays. . We have completed the present invention by optimizing three sensitive and specific real-time PCR assays useful for the detection and quantification of respiratory specimens for infectious and pathogenic studies on HBoV.

In other words, the present invention provides a method for detecting bocavirus (HBoV) by detecting NS1, NP-1 and VP1 genes of bocavirus by real-time PCR method, wherein the sequence number is used as a potential primer for NS1 gene detection. : Base sequence represented by SEQ ID NO: 1, base sequence represented by SEQ ID NO: 2 as an inverted primer, and base sequence represented by SEQ ID NO: 3 as a probe; A base sequence represented by SEQ ID NO: 4 as a translocation primer, a base sequence represented by SEQ ID NO: 5 as an inversion primer, and a base sequence represented by SEQ ID NO: 6 as a probe for NP-1 gene detection; And a nucleotide sequence represented by SEQ ID NO: 7 as a translocation primer, a nucleotide sequence represented by SEQ ID NO: 8 as an inversion primer, and a nucleotide sequence represented by SEQ ID NO: 9 as a probe for the VP1 gene detection.

The present invention also provides a method for producing a nucleic acid, comprising the steps of: (1) obtaining a nucleic acid from a clinical sample; (2) performing real-time PCR using the nucleic acid obtained in step (1) using the nucleotide sequences represented by SEQ ID NO: 1 to SEQ ID NO: 9 as primers and probes; And (3) identifying the NS1, NP-1 and VP1 genes of the bocavirus from the PCR product of step (2).

The present invention also provides a kit for detecting a bocavirus comprising primers and probes for detecting NS1, NP-1 and VP1 genes of bocavirus by real-time PCR, wherein the primers and probes are SEQ ID NO: 1 to SEQ ID NO: : A nucleotide sequence represented by 9.

 Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Materials and methods

Clinical sample

A nasal aspirates clinical sample was taken from a pediatric patient with acute respiratory tract infection in a domestic clinical hospital. The sampling was acute from the Korea Centers for Disease Control (KCDC) between January 2006 and January 2007. It was conducted under respiratory infection surveillance. Sampling was performed with parental consent of the child. We used 102 respiratory samples collected in winter between 2006 and 2007. Within 24 hours after sampling, total nucleic acids were extracted and stored at −70 ° C. using an automated MagNa pure LC (Roche Diagnostic, Germany) total nucleic acid extraction kit from 200 ul of each sample.

Primer and Probe Design

For primer and probe design, the entire HBoV sequence was obtained from the ST1 strain (GeneBank accession no, DQ000495). HBoV NS1, NP-1 and VP1 genes were searched to find target sites applicable to TaqMan real-time PCR using Primer Express software version 2.0 (Applied Biosystems, Foster City, CA, USA) . Applied Biosystems (Foster City, CA, USA) synthesized optimal primer and probe sets with no major nonspecific homology and minimal likelihood of double or hairpin formation. For TaqMan MGB (minor groove binder) probes, the 5 'end was labeled with 6-carboxyfluorescein (FAM) and the 3' end was labeled with Black Hole Quencher (Table 1).

Table 1. Primers and Probes Used for Real-Time PCR Assays for HBoV

^a nucleotide position is HBoV strain ST1 (GeneBank accession no, DQ000495).

Composition of Plasmid DNA Standards

Approximately 5.2 kbp of the HBoV whole genome was transferred to a translocation primer (5 'GGA TTC CAA GAT GGC GTC TGT 3', SEQ ID NO: 10) and inverted primer (5 'ACA CAT TAA AAG ATA TAG AGT TTC 3', SEQ ID NO: 11) Was amplified from the PCR positive sample. The result was confirmed by cloning and sequencing in the plasmid vector pCR2.1-TOPO (Invitrogen, USA). The obtained pCR2.1-HBoV plasmid was purified by QIamp midi prep kit (QIAGEN, Germany) and quantified using Qubit ^™ fluorometer (Invitrogen, USA). Quantification was performed twice and converted to number of molecules (5 × 10 ⁸ DNA copies / μl). For standard curve generation, 10-fold series dilutions of pCR2.1-HBoV plasmids were prepared in 10 mM Tris-EDTA buffer (pH 8.0) and stored at -20 ° C.

Real time analysis of human bocavirus

10 μl TaqMan ^® Universal PCR Master Mix (Applied Biosystems, USA) containing 2 μl DNA extract or standard plasmid, ROX as passive reference dye, and 900 nM potential and reverse primer and 250 Real-time PCR was performed in 20 μl reaction mixture containing nM probes. The amplification and detection was carried out under the following conditions from the ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA ): uracil - N - glycoside as active silanol claim 2 minutes at 50 ℃ (uracil- N -glycosylase, UNG ) , Then perform PCR for 10 minutes at 95 ° C and amplify 40 cycles (15 seconds at 95 ° C, 1 minute at 60 ° C). In order to normalize the signal for non-PCR-related fluorescence waves occurring between wells, measurement of reporter staining (FAM) signal against internal inactive staining (ROX) signal was performed. Cycle inverse ( C _T ) means the number of cycles beyond the fixed limit non-template control value (NTC). The assay for each assay was Sequence Sequencer Software Ver. 2.1 (Applied Biosystems, USA). Positive values were determined to be positive for all three target genes (NS1, NP-1 and VP1) in excess of 10 copy numbers.

Comparative example

Conventional PCR Methods for Human Bacovirus

To compare the sensitivity of real-time PCR, conventional PCR was performed by using the following primer sets and amplifying 346 bp fragments of the NS1 gene (640-986 bp). Primer sets: F3 (5'-TGG CTA CAC GTC CTT TTG AAC C 3 ', SEQ ID NO: 12) and R2 (5'- GAC TTC GTT ATC TAG GGT TGC G-3', SEQ ID NO: 13). 5 μL DNA extract, 5 μl 10 × PCR buffer, 2 μl dNTP mixture (final concentration of each dNTP is 400 μM), 1 μl SP Taq (COSMO GENTEC, Korea), 1 μl of each primer (10pM), and nuclease PCR analysis was performed with 50 μl reaction volume containing free distilled water. The PCR reaction was carried out for 15 minutes at 95 ° C., 35 cycles amplification (30 seconds at 94 ° C., 30 seconds at 55 ° C., 30 seconds at 72 ° C., and 30 seconds at 72 ° C.). CA, USA) and used at 72 ° C. for 10 minutes. Deployed to gel the PCR product on 2% agarose and stained with ^{SYBR ® safe DNA gel stain dye (} Invitrogen, USA) and it was visually observed under a UV light.

Example

Example 1: Real-Time PCR Standard Curves and Dynamic Ranges

To confirm the absolute amount of viral load, standard curves were generated using HBoV plasmid DNA. Ten-fold series dilutions were used as reaction mixtures from 50 to 5 × 10 ⁷ plasmid copies, and each reaction was performed three times. The standard curve generated in each analysis showed amplification efficiency (NS1, 96.8%; NP-1, 89.5%; VP1, 104.8%) and linear regression of about 0.99 in three analyzes (FIG. 1A). Analytical dynamic range was analyzed using C _T values under 7 log units. Corresponding C _T Values range from 35.48 ± 0.4 for 50 copies to 13.4 ± 0.02 for 5 × 10 ⁷ copies for the NS1 gene; For the NP-1 gene, ranging from 36.51 ± 0.36 for 50 copies to 13.7 ± 0.06 for 5 × 10 ⁷ copies; And for the VP1 gene, from 35.82 ± 0.2 for 50 copies to 13.83 ± 0.03 for 5 × 10 ⁷ copies (FIG. 1B). In FIGS. 1A and 1B, 10-fold series dilutions of 50-5 × 10 ⁷ clones of the pCR2.1-HBoV plasmid were tested three times for each assay. C _T Standard curve graphs were made by applying values to the y axis and the amount of DNA introduced on the x axis. Tilt values (NS1, -3.4; NP-1, -3.2; VP1, -3.61) and correlation co-factor determination ( R ² ) (NS1, 0.99; NP-1, 0.99; VP1, 0.99) were added to each assay Shown (FIG. 1A). The dynamic range of each assay ranges from 35.48 ± 0.4 to 5 x 10 ⁷ copies for 50 copies in the NS1 gene and 13.4 ± 0.02 for 50 copies for the NP-1 gene and 36.51 ± 0.36 to 5 x 10 ⁷ copies for 50 copies for the NP-1 gene. 13.7 ± 0.06, and expanded to 7 log units, with corresponding C _T values ranging from 35.82 ± 0.2 to 5 x 10 ⁷ copies for 50 copies for the VP1 gene, and 13.83 ± 0.03 for 5 x 10 ⁷ copies (FIG. 1B). Normalized reporter (Rn) is the fluorescence emission intensity of reporter staining divided by passive reference dye intensity, and ΔRn means Rn excluding Rn without reaction in the reaction sample.

Example 2: Real-Time PCR Detection Limits, Sensitivity and Reproducibility

Assay detection limits were determined in 12 replicate experiments using each primer / probe set with 10, 5 and 1 copies of plasmid DNA. The detection rates of 10 copies were 100% (NP-1), 100% (NS1) and 100% (VP1). At 5 copies, the assay detection limit was reduced to 75% (NP-1), 75% (NS1) and 25% (VP1). At the lowest level of template (1 copy), NP-1 gene (53.3%) was higher than other target genes NS1 (33.3%) and VP1 (6.7%). Other DNA viruses such as human parvovirus B19, adenoviruses, herpes simplex viruses 2 and 16, and varicellazoster viruses, and respiratory pathogenic bacteria such as Heamophilus Influenzae , Legionella (Legionella), were evaluated for cross-reactivity with pneumoniae (S. pneumonia, M. pneumonia and chlamydia pneumonia). In addition, respiratory RNA viruses (influenza viruses A, B, parainfluenza viruses 1, 2 and 3, respiratory syncytial virus, rhinovirus and CDNA of enterovirus} was used. No non-specific amplification of these samples was detected in each assay. In assay, inter assay reproducibility was measured three times a day with 10-fold serial dilutions of standard HBoV plasmid DNA copies ranging from 50 to 5 × 10 ⁸ copies. Mean C _T , standard deviation (SD) and covariate (CV) were estimated. Intravariate variation of 0.03% to 1.12% and interanalysis variation of 0.03% to 3.43% indicate high assay reproducibility precision (Table 2).

Table 2. Reproducibility in and between assays of HBoV real-time PCR ^a

^A 10-fold dilution of HBoV plasmid DNA was analyzed three times and three days. The C _T mean values, standard deviations (SDs) and variance cavity coefficients (CVs) were calculated.

^b Each value measured from three replicates in one analysis

^c three independent analyzes on different days

The characteristics of HBoV real-time PCR positive patients are shown in Table 3.

Table 3. Characteristics of 5 HBoV Real-Time PCR Positive Patients

^* Average viral load per ml of sample in 3 pairs of primer / probe sets, calculated in 3 replicate experiments

Example 3: Comparison of Real-Time PCR and Conventional PCR Assays

To determine the sensitivity of the real-time PCR of the present invention compared to conventional PCR, a 10-fold serial dilution of standard HBoV plasmid DNA copies ranging from 100 to 1 × 10 ⁸ copies was used as template (FIG. 2). In Figure 2, Lane M is 100bp marker (Invitrogen, USA); Lanes 1-7, 10-fold serial dilution of HBoV plasmid standard; Lane 8 represents the negative control. 2 μl of HBoV plasmid standard DNA (5 × 10 ⁸ DNA copies / μl) was used in each assay. The limit of detection of the gel based on conventional PCR analysis of amplifying 346 bp NS1 gene was 100 copies of HBoV lasmid DNA. In contrast, three real-time PCR analyzes yielded detection limits from plasmid DNA samples containing 10 copies. Figure 3 is a comparison of the mean C _T value in the positive clinical specimens analyzed three times.

Example 4: Kit

Using the method for detecting the NS1, NP-1 and VP1 genes of bocavirus according to the present invention, the present invention provides a kit for detecting bocaviruses comprising primers and probes for screening and detecting the genes of bocaviruses. do. The primers and probes are described in Table 1 above.

As such, three highly sensitive and specific real-time assays for HBoV diagnosis and quantitation can be usefully used for infectious and pathogenic studies of HBoV.

<110> Korea Center for Disease Control and Prevention <120> Real-time PCR assays for absolute Quantification for Human          Bocavirus <130> IPM-33408 <160> 13 <170> KopatentIn 1.71 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for NS1 <400> 1 tgaacctgaa gaggctgaca tatttc 26 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for NS1 <400> 2 catgttgccg ccagtaactc 20 <210> 3 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Probe for NS1 <400> 3 caccccatgc ctctcg 16 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for NP-1 <400> 4 cagccaccta tcgtcttgca 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for NP-1 <400> 5 ccctcgtctt catcacttgg t 21 <210> 6 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Probe for NP-1 <400> 6 ctgcttcgaa gacctc 16 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for VP1 <400> 7 agctgtcact tctcaccaca ag 22 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for VP1 <400> 8 ttgctttagg tctgaagcgc ttat 24 <210> 9 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Probe for VP1 <400> 9 attggcagcg ccttac 16 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 10 ggattccaag atggcgtctg t 21 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 11 acacattaaa agatatagag tttc 24 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> F3 primer <400> 12 tggctacacg tccttttgaa cc 22 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> R2 primer <400> 13 gacttcgtta tctagggttg cg 22

Claims (4)

A method of detecting bocavirus (HBoV) by detecting NS1, NP-1 and VP1 genes of bocaviruses by real-time PCR, wherein the nucleotide sequence represented by SEQ ID NO: 1 as a reverse primer for NS1 gene detection A base sequence represented by SEQ ID NO: 2 as a probe and a base sequence represented by SEQ ID NO: 3 as a probe; A base sequence represented by SEQ ID NO: 4 as a translocation primer, a base sequence represented by SEQ ID NO: 5 as an inversion primer, and a base sequence represented by SEQ ID NO: 6 as a probe for NP-1 gene detection; And a nucleotide sequence represented by SEQ ID NO: 7 as a translocation primer, a nucleotide sequence represented by SEQ ID NO: 8 as an inversion primer, and a nucleotide sequence represented by SEQ ID NO: 9 as a probe for detection of the VP1 gene. How to detect a virus. (1) obtaining nucleic acid from a clinical sample; (2) performing real-time PCR using the nucleic acid obtained in step (1) using the nucleotide sequences represented by SEQ ID NO: 1 to SEQ ID NO: 9 as primers and probes; And (3) identifying the NS1, NP-1 and VP1 genes of bocavirus from the PCR product of step (2). A kit for detecting a bocavirus, comprising a primer and a probe of the nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 9 for detecting NS1, NP-1 and VP1 genes of bocavirus by real-time PCR. delete

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