Dual-labeled Probe Research Articles

TaqMan reverse transcription polymerase chain reaction for the detection of Japanese encephalitis virus

One step TaqMan reverse transcription polymerase chain reaction (RT-PCR) using TaqMan probe was developed for detection of Japanese encephalitis virus (JEV). Real-time RT-PCR was optimized to quantify JEV using the detection system (Rotor Gene 2000 detector) and dual-labeled fluorogenic probes. The gene specific labeled fluorogenic probe for the 3' non-translated region (3' NTR) was used to detect JEV. When the specificity of the assay using specific JEV primers was evaluated by testing three different JEV strains, other swine viruses and bovine viral diarrhea virus, no cross-reactions were detected with non-JE reference viruses. A single tube TaqMan assay was shown to be 10-fold more sensitive than the conventional two-step RT-PCR method. Detection limits of two step and real-time RT-PCR for JEV were 112 TCID(50) /ml and 11.2 TCID(50) /ml, respectively. Quantification of JEV was accomplished by a standard curve plotting cycle threshold values (C(t)) versus infectivity titer. Real-time RT-PCR assay using single tube method could be used as a sensitive diagnostic test, and supplied the results in real time for detection and quantification of JEV. We could detect JEV RNA genome in plasma samples of pigs inoculated with KV1899 strain at 2 days post inoculation, but couldn't in 41 fetus samples. This assay was sensitive, specific, rapid and quantitative for the detection of JEV from laboratory and field samples.

Single-nucleotide polymorphism genotyping by melting analysis of dual-labeled probes: examples using factor V Leiden and prothrombin 20210A mutations.

The emergence of fluorescence techniques in 96- or 384-well formats has led to high-throughput single-nucleotide polymorphism (SNP) detection methods. Among these, allele discrimination based on real-time PCR technologies has been developed along two main methodologic approaches. In the first approach, a temperature-dependent fluorescent signal is generated by hybridization of a probe at the end of each PCR cycle. The monitoring of fluorescence emission is followed during (1)(2) or at the end of (3)(4) the PCR process. In the latter case, allele detection is achieved by carrying out of a melting curve analysis. In the second approach, exploiting the classic 5′ nuclease assay (TaqMan®) technology, a temperature-independent fluorescence signal is generated during the PCR process by the exonuclease degradation of a hybridized TaqMan probe (5)(6). Except for one method discussed later (3), all of these techniques require the use of two probes for allele differentiation. An allele differentiation study has stressed the risk of artifacts and subsequent erroneous results when a polymorphism is located within the DNA sequence targeted by TaqMan (7). The same study demonstrated the power of methods relying on determination of melting curves. Although other interesting approaches have been reported recently, such as DASH (8), iFret (9), and dipole calculation (10), some of them are technically difficult or time-consuming. In addition, the SYBR Green technique (11) would represent the simplest method to detect point mutations, but it seems to be limited by its lack of ability to differentiate alleles (12). We present here a new methodologic approach that allows high-throughput genotyping of SNPs. Allele differentiation is achieved by the generation of melting curves, with a single dual-labeled probe, at the end of a classic PCR reaction. The reliability of the method is demonstrated for detection of the factor V Leiden …

Covalent Histone Modifications Underlie the Developmental Regulation of Insulin Gene Transcription in Pancreatic β Cells

Histone modifying enzymes contribute to the activation or inactivation of transcription by ultimately catalyzing the unfolding or further compaction, respectively, of chromatin structure. Actively transcribed genes are typically hyperacetylated at Lys residues of histones H3 and H4 and hypermethylated at Lys-4 of histone H3 (H3-K4). To determine whether covalent histone modifications play a role in the beta cell-specific expression of the insulin gene, we performed chromatin immunoprecipitation assays using anti-histone antibodies and extracts from beta cell lines, non-beta cell lines, and ES cells, and quantitated specific histone modifications at the insulin promoter by real-time PCR. Our studies reveal that the proximal insulin promoter is hyperacetylated at histone H3 only in beta cells. This hyperacetylation is highly correlated to recruitment of the histone acetyltransferase p300 to the proximal promoter in beta cells, and is consistent with the role of hyperacetylation in promoting euchromatin formation. We also observed that the proximal insulin promoter of beta cells is hypermethylated at H3-K4, and that this modification is correlated to the recruitment of the histone methyltransferase SET7/9 to the promoter. ES cells demonstrate a histone modification pattern intermediate between that of beta cells and non-beta cells, and is consistent with their potential to express the insulin gene. We therefore propose a model in which insulin transcription in the beta cell is facilitated by a unique combination of transcription factors that acts in the setting of an open, euchromatic structure of the insulin gene.

CARBOHYDRATE INGESTION INFLUENCES SKELETAL MUSCLE CYTOKINE mRNA AND PLASMA CYTOKINE LEVELS AFTER A 3-H RUN

PURPOSE To determine the influence of carbohydrate ingestion on skeletal muscle cytokine mRNA content and plasma cytokine levels after a 3-h run. METHODS Sixteen experienced marathoners ran on treadmills for 3 h at ∼70% VO2max on two occasions while receiving 1 l/h carbohydrate (CHO) or placebo (PLA) beverages. Blood and vastus lateralis muscle biopsy samples were collected before and after exercise. Plasma was analyzed for IL-6, IL-10, IL-1ra, IL-8, cortisol, glucose, and insulin. Muscle was analyzed for glycogen content and relative gene expression of 13 cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p35, IL-12p40, IL-15, IFN-γ, TNF-α) using real-time quantitative RT-PCR and the ΔΔCt method that monitors the degradation of a dual-labeled fluorescent probe (TaqMan) in real time concomitant with PCR amplification. The data were analyzed using a 2 × 2 repeated measures ANOVA. RESULTS Plasma glucose and insulin were higher and cortisol, IL-6, IL-10, and IL-1ra, but not IL-8, were significantly lower post-exercise in CHO vs PLA. Change in muscle glycogen content did not differ between CHO and PLA (P=0.246). Muscle cytokine mRNA was detected for IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p35, IL-15, IFN-γ, and TNF-α, but only IL-1β, IL-6, and IL-8 were significantly increased with exercise (P < 0.05). The increase in mRNA (fold difference from pre-exercise) was attenuated in CHO (15.9-fold) compared to PLA (35.2-fold) for IL-6 (P = 0.071) and IL-8 (CHO, 7.8-fold; PLA, 23.3-fold; P = 0.063). CONCLUSION CHO compared to PLA beverage ingestion attenuates the increase in plasma IL-6, IL-10, and IL-1ra, and gene expression for IL-6 and IL-8 in athletes running 3 h at 70% VO2max despite no differences in muscle glycogen content. Supported by a grant from the Gatorade Sports Science Institute.

Quantitative detection of agar-cultivated and rhizotron-grown Piloderma croceum Erikss. & Hjortst. by ITS1-based fluorescent PCR.

A real-time quantitative TaqMan-PCR was established for the absolute quantification of extramatrical hyphal biomass of the ectomycorrhizal fungus Piloderma croceum in pure cultures as well as in rhizotron samples with non-sterile peat substrate. After cloning and sequencing of internal transcribed spacer (ITS) sequences ITS1/ITS2 and the 5.8S rRNA gene from several fungi, including Tomentellopsis submollis, Paxillus involutus, and Cortinarius obtusus, species-specific primers and a dual-labelled fluorogenic probe were designed for Piloderma croceum. The dynamic range of the TaqMan assay spans seven orders of magnitude, producing an online-detectable fluorescence signal during the cycling run that is directly related to the starting number of ITS copies present. To test the confidence of the PCR-based quantification results, the hyphal length of Piloderma croceum was counted under the microscope to determine the recovery from two defined but different amounts of agar-cultivated mycelia. Inspection of the registered Ct values (defined as that cycle number at which a statistically significant increase in the reporter fluorescence can first be detected) in a 10-fold dilution series of template DNA represents a suitable and stringent quality control standard for exclusion of false PCR-based quantification results. The fast real-time PCR approach enables high throughput of samples, making this method well suited for quantitative analysis of ectomycorrhizal fungi in communities of natural and artificial ecosystems, so long as applicable DNA extraction protocols exist for different types of soil.

Synthesis of HyBeacons and dual-labelled probes containing 2'-fluorescent groups for use in genetic analysis.

An FMOC-protected 2'-hydroxyethyl uridine phosphoramidite has been used to synthesise fluorescein-labelled HyBeacon probes and "FAM-ROX" dual-labelled fluorogenic oligonucleotides.

Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

BackgroundReal-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive.ResultsWe have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy.ConclusionOur assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

Photoluminescence and electrochemiluminescence of a Ru(II)(bpy)3-quencher dual-labeled oligonucleotide probe.

A molecular beacon oligonucleotide probe covalently labeled with Ru(II)(bpy)3 and Black Hole Quencher-2 is synthesized, and hybridization assays are performed using photoluminescence and electrochemiluminescence methods of excitation.

Developmental changes in cyclo-oxygenase mRNA induction by hypoxia in rat kidney.

Prostaglandins, synthesized by cyclo-oxygenase (COX), regulate renal hemodynamics and also epithelial water and solute transport. Developmental changes occur in COX-2 mRNA expression and its response to lipopolysaccharide stimulation in rats. We examined age-related changes in COX mRNA expression induced by hypoxia in the renal cortex and medulla of developing rats. Total RNA was extracted from 1- and 4-week-old male Wistar rats exposed to one or 4 h of hypoxia (8% O2). Cyclo-oxygenase mRNA was quantitatively analyzed using a real-time polymerase chain reaction with dual-labeled fluorogenic probes. Expression of COX-1 mRNA did not change in response to hypoxia in the cortex or medulla in either infantile or adult rats. In infantile rats, COX-2 mRNA expression was not induced by one or 4 h of hypoxia. In adults, 1- and 4-h exposures to hypoxia induced COX-2 mRNA in the renal cortex, and 1-h of exposure induced COX-2 mRNA in the medulla. Response of expression of COX-2 mRNA in rats exposed to acute hypoxia show age-related variability treated by acute hypoxia. Cyclo-oxygenase-2 might not play a major role in the hypoxic infantile rat kidney.

Transcription and expression of CD44 variant exons by oro-pharyngeal squamous cell carcinomas

The CD44 cell surface hyaluronan binding protein has many isoforms formed by alternative splicing and glycosylation, and some variants have been associated with a malignant phenotype, invasion and metastasis. CD44 splicing patterns and intron 9 retention in 24 oro-pharyngeal squamous cell carcinomas (OSCC) were investigated by RT-PCR. Transcription efficiency and quantity were determined in 10 carcinomas and normal control mucosa by real-time PCR using dual-labeled fluorescent Taqman probes. Most of the carcinomas, regardless of grade, showed one major transcript including exons v2-v10, similar to that expressed by normal keratinocytes. In addition, most carcinomas expressed a variety of truncated transcripts of contiguous variant exons. Real-time PCR revealed that carcinomas showed both over-expression and down-regulation in transcription compared to normal keratinocytes, but this change was independent of the state of differentiation or the sub-site of biopsy. Intron 9 was not retained in normal keratinocytes or carcinomas. Overall, the results indicate that OSCC, like normal keratinocytes, constitutively express all variant exons and that the missplicing seen in other malignancies does not appear to occur. OSCC do not reproducibly over-express CD44 and show a wide range of transcription levels. CD44 expression does not appear to be fundamentally deranged in OSCC and is unlikely to be of value in early diagnosis, or as a prognostic marker.

Fluorescent multicolor multiplex homogeneous assay for the simultaneous analysis of the two most common hemochromatosis mutations

We report the development of a qualitative fluorescent multiplex homogeneous assay designed for the detection of the two most common hemochromatosis mutations using dual-labeled fluorescent probes. The assay is able to detect four allelic variants in a single closed tube using a single thermocycling protocol. The procedure combines the great sensitivity of the polymerase chain reaction, the specificity provided by allele-specific oligonucleotide hybridization using the 5 ′ nuclease assay format, and the higher throughput of a multicolor fluorescence detection procedure. Genomic DNA was prepared from whole blood specimens using standard procedures. Following DNA sample preparation, two regions of the hemochromatosis gene (HFE) including the H63D and C282Y mutations were coamplified and detected in real-time by four different fluorescently labeled allele-specific oligonucleotide probes. Assay specificity was demonstrated by a blind methods comparison study that included 37 DNA samples from individuals with a known HFE genotype. Results from the study showed that the multicolor multiplex HFE assay unambiguously classified all possible genotypes for the HFE gene C282Y and H63D mutations 1 See, e.g., U.S. Patents 5,712,098 and 6,025,130. 1 . This technique will be useful for research and molecular diagnostic laboratories and can be easily adapted for the detection of other single nucleotide polymorphisms.

Intramolecular dimers: a new strategy to fluorescence quenching in dual-labeled oligonucleotide probes.

Many genomics assays use profluorescent oligonucleotide probes that are covalently labeled at the 5' end with a fluorophore and at the 3' end with a quencher. It is generally accepted that quenching in such probes without a stem structure occurs through Förster resonance energy transfer (FRET or FET) and that the fluorophore and quencher should be chosen to maximize their spectral overlap. We have studied two dual-labeled probes with two different fluorophores, the same sequence and quencher, and with no stem structure: 5'Cy3.5-beta-actin-3'BHQ1 and 5'FAM-beta-actin-3'BHQ1. Analysis of their absorption spectra, relative fluorescence quantum yields, and fluorescence lifetimes shows that static quenching occurs in both of these dual-labeled probes and that it is the dominant quenching mechanism in the Cy3.5-BHQ1 probe. Absorption spectra are consistent with the formation of an excitonic dimer, an intramolecular heterodimer between the Cy3.5 fluorophore and the BHQ1 quencher.

QuantifyingAphanomyces euteichesin Alfalfa with a Fluorescent Polymerase Chain Reaction Assay

ABSTRACT A polymerase chain reaction (PCR) assay using a set of specific primers and a dual-labeled probe (TaqMan) was developed to quantify the amount of Aphanomyces euteiches DNA in alfalfa plants exhibiting varying levels of disease severity. The study included isolates of race 1 and race 2 of A. euteiches. The assay also discriminated between alfalfa populations for resistance based on analysis of DNA extracted from bulked plant samples. Analysis of individual plants and bulked plant samples of standard check populations with both pathogen isolates resulted in Spearman rank correlations between pathogen DNA content and disease severity index ratings that were greater than 0.75 and highly significant (P < 0.0005). In experiments with a race 1 isolate, the amount of pathogen DNA present in the resistant check WAPH-1 was significantly less than in the susceptible check Saranac. In experiments with a race 2 isolate, the amount of pathogen DNA in the resistant check WAPH-5 was significantly less than in either of the susceptible checks, Saranac and WAPH-1. Discrimination between commercial cultivars based on quantitative PCR analysis of bulked plant samples was similar to classification based on visual assessment of disease severity.

Quantification and duration of viraemia during hepatitis A infection as determined by real-time RT-PCR.

Human transmission studies and molecular techniques have provided evidence that transient viraemia occurs during infection with hepatitis A virus (HAV). However, the duration of its presence and levels during the phases of clinical disease and convalescence has not yet been well studied in human patients. Real-time RT-PCR techniques are increasingly used to quantify RNA viruses for diagnosis and/or research purposes. We have optimized a one-step RT-PCR that contains a dual-labelled fluorogenic probe to quantify the 5' noncoding region (5' NCR) of HAV. This method has a dynamic range (5-5 x 10(6) copies). The coefficient of regression of the standard curve was, on average 0.978. Intra-assay CVs% varied from 6.1% to 0.98%, and interassay CVs% from 6.46% to 2.1%. In the currently reported study 41 HAV IgM positive serum samples and 200 serum samples from healthy blood donors were tested by the quantitative RT-PCR method. The mean values on the first day of diagnosis found was 6.38 x 10(5) copies/mL. In a longitudinal study, viraemia persisted for an average of 60 days after clinical onset. These results show that viraemia in HAV infection lasts for many weeks.

Developmental changes in cyclooxygenase mRNA expression in the gastric mucosa of rats.

In newborn rats, gastric mucosa is more susceptible to various damaging agents and recovers from injury more quickly than in older animals. To determine whether metabolism of prostaglandins is responsible for this mucosal protective mechanism in developing rats, we studied cyclooxygenase (COX) mRNA expression in the mucosa using quantitative real-time polymerase chain reaction (PRC). Cyclooxygenase-1 and COX-2 mRNA was extracted from the gastric mucosa of rats of various ages and quantitatively analyzed using real-time PCR with dual-labeled fluorogenic probes. The copy numbers of cDNA for COX-1 and COX-2 were standardized to glyceraldehyde-3-phosphate dehydrogenase from the same sample. Cyclooxygenase-1 mRNA expression was lowest in 1-week-old rats and highest in 4-week-old rats. Mucosal damage produced by 150 mmol/L HCl and 60% ethyl alcohol did not increase COX-1 mRNA expression in any age group. Cyclooxygenase-2 mRNA expression increased significantly with age. Mucosal injury increased COX-2 mRNA in each age group, especially in 1-week-old rats. Intraperitoneal lipopolysaccharide also increased COX-2 mRNA in both 1- and 4-week old rats. The high level of COX-2 mRNA expression in the gastric mucosa of 1-week-old rats may be responsible for the physiologic characteristics of gastric mucosal defenses in this age group.

Fluorogenic RT–PCR assay (TaqMan) for detection and classification of bovine viral diarrhea virus

A single tube fluorogenic RT–PCR-based ‘TaqMan’ assay was developed for detection and classification of bovine viral diarrhea virus (BVDV). TaqMan–PCR was optimized to quantify BVD virus using the ABI PRISM 7700 sequence detection system and dual-labeled fluorogenic probes. Two different gene specific labeled fluorogenic probes for the 5′ untranslated region (5′ UTR) were used to differentiate between BVD types I and II. Sensitivity of the single tube TaqMan assay was compared with two-tube TaqMan assay and standard RT–PCR using 10-fold dilutions of RNA. Single tube TaqMan assay was 10–100-fold more sensitive than the two-tube TaqMan assay and the standardized single tube RT–PCR. Specificity of the assay was evaluated by testing different BVD virus strains and other bovine viruses. A total of 106 BVD positive and negative pooled or single serum samples, field isolates and reference strains were tested. Quantitation of cRNA from types I and II BVD virus was accomplished by a standard curve plotting cycle threshold values ( C T) versus copy number. Single tube TaqMan–PCR assay was sensitive, specific and rapid for detection, quantitation and classification of BVD virus.

Developmental changes in cyclooxygenase mRNA expression in the kidney of rats.

Prostaglandins, synthesized by cyclooxygenase (COX), regulate renal hemodynamics and also epithelial water and solute transport. To determine whether COX mRNA expression changes with age, we studied expression in renal medulla and in cortex in developing rats at various ages. We also examined age-related changes in COX mRNA expression induced by lipopolysaccharide (LPS). COX mRNA was quantitatively analyzed in a real-time polymerase chain reaction (PCR) with dual-labeled fluorogenic probes. COX-1 mRNA expression did not change with age in cortex or medulla. COX-2 mRNA expression was highest in 1-week-old rats and lowest in 4- and 8-week-old rats. Lipopolysaccharide treatment did not alter COX-1 mRNA expression in infantile or adult rats. In adults, LPS at 1, 5, and 10 mg/kg induced COX-2 mRNA expression in renal medulla; the higher doses, 5 and 10 mg/kg, induced COX-2 expression in cortex. In infantile rats, COX-2 mRNA, already high in the unmanipulated state, was further increased by only 1 mg/kg LPS in both renal cortex and medulla. Age-related changes in the expression of COX-2 mRNA might be responsible for changing physiologic characteristics of renal function during postnatal development in rats, and may be important in renal cortical development.

Type-2 somatostatin receptor mRNA levels in breast and colon cancer determined by a quantitative RT-PCR assay based on dual label fluorogenic probe and the TaqMan™ technology

We reported previously that the expression of type 2 somatostatin receptor (sst2) was positively related to patient outcome in the childhood tumor neuroblastoma. To quantitate the expression of mRNA sst2 expression, we used a competitive RT-PCR assay. To improve the practicability of this measurement and its applicability to large groups of patients, we present here an original ‘real-time’ quantitative RT-PCR method, based on a dual-labeled fluorogenic probe and the TaqMan™ technology. By this method, we have measured sst2 mRNA expression in 24 breast cancer samples and 26 colon carcinomas as well as on the corresponding non-adjacent non-neoplastic tissue of the same patients. The proposed method has a dynamic range of 4×10 4 to 4×10 8 molecules of sst2 mRNA. The intra-assay precision of the test, evaluated as signal detection variability, was 2.4%. Accuracy, evaluated by the addition of standard RNA to unknown samples, provided a mean recovery of 98±2%. A significant correlation has been observed in a study performed in 24 neuroblastoma samples measured both with the proposed method and with a competitive RT-PCR assay ( r=0.913, p<0.001). In our preliminary clinical study, no significant differences were observed in sst2 mRNA levels between normal and tumor specimens in both colorectal (normal tissue 5.1×10 7±2.0×10 7 molecules/μg total RNA, cancer tissue 9.7×10 7±4.2×10 7) and breast tumors (normal tissue 5.5×10 8±2.0×10 8, cancer tissue 4.4×10 8±3,7×10 8). However, in colorectal cancer, sst2 mRNA values of subjects with high circulating carcinoembryonic antigen (CEA) levels (>5 ng/ml) were statistically lower (2.3×10 7±6.2×10 6 molecules/μg total RNA; p<0.05) than those of subjects with low CEA concentration (1.4×10 8±6.7×10 7). Also, the sst2 mRNA ratio between normal and tumor tissue (N/T ratio) resulted significantly inversely related to CEA levels. In breast cancer, a significant difference was found between the mean N/T ratio of negative (below 10 fmol/mg protein) and positive estrogen receptor tumors ( p<0.05). Analogous results were found selecting breast tumors on the basis of the progesterone receptor status ( p<0.05). The proposed method is accurate, precise, sensitive and less labor-intensive than the competitive RT-PCR assay. For a correct evaluation of sst2 mRNA expression, it seems very important to measure the sst2 expression both in tumor and in the non-tumoral non-adjacent tumor specimens.

二重標識蛍光プローブを用いたHLA-BアリルのリアルタイムPCR-SSP法の開発

PCR-SSP法と二重標識蛍光プローブとを組み合わたリアルタイムPCR-SSP法は, ABIPRISM(TM)7700シーケンスディテクターを利用して, リアルタイムにPCR産物を測定することで, PCR-SSP増幅後の付加的操作なしに短時間でアリルが決定できるシステムである. 我々は, このシステムを利用することがもっとも困難と思われるHLA-Bアリルに応用し, その実用性と問題点について検討した. HLA-B抗原の特異性が既知の34試料を対象とし, 市販のSSPプライマーキットと我々が設計した7種類の二重標識蛍光プローブを利用して行った. すべてのサンプルは, PCR増幅を35回行うことでもっとも良好な反応態度を示した. プライマーの特異性から増幅が期待されるサンプルはすべてΔRn閾値以上で, 陰性は閾値未満であり, 偽反応はみられなかった. また, 各サンプルでもっとも弱かった陽性ΔRn値と強かった陰性ΔRn値の比は6. 18であり, 判定上問題となることはなかった. 今回開発したリアルタイムPCR-SSPHLA-Bタイピングシステムで判定されたアリルタイプはパネルDNAの血清学的特異性とまったく矛盾しなかった. また, DNA抽出からHLA-Bアリル同定までの過程を約2時間で終了することができた. この方法はPCR増幅から反応結果出力までの過程が自動化されるているので人為的ミスを最小限に抑えることができるものと考えられた.

Real-time Quantitative PCR for Retrovirus-like Particle Quantification in CHO Cell Culture

Chinese hamster ovary (CHO) cells have been widely used to manufacture recombinant proteins intended for human therapeutic uses. Retrovirus-like particles, which are apparently defective and non-infectious, have been detected in all CHO cells by electron microscopy (EM). To assure viral safety of CHO cell-derived biologicals, quantification of retrovirus-like particles in production cell culture and demonstration of sufficient elimination of such retrovirus-like particles by the down-stream purification process are required for product market registration worldwide. EM, with a detection limit of 1×106particles/ml, is the standard retrovirus-like particle quantification method. The whole process, which requires a large amount of sample (3–6 litres), is labour intensive, time consuming, expensive, and subject to significant assay variability. In this paper, a novel real-time quantitative PCR assay (TaqMan assay) has been developed for the quantification of retrovirus-like particles. Each retrovirus particle contains two copies of the viral genomic particle RNA (pRNA) molecule. Therefore, quantification of retrovirus particles can be achieved by quantifying the pRNA copy number, i.e. every two copies of retroviral pRNA is equivalent to one retrovirus-like particle. The TaqMan assay takes advantage of the 5′→3′ exonuclease activity of Taq DNA polymerase and utilizes the PRISM™ 7700 Sequence Detection System of PE Applied Biosystems (Foster City, CA, U.S.A.) for automated pRNA quantification through a dual-labelled fluorogenic probe. The TaqMan quantification technique is highly comparable to the EM analysis. In addition, it offers significant advantages over the EM analysis, such as a higher sensitivity of less than 600particles/ml, greater accuracy and reliability, higher sample throughput, more flexibility and lower cost. Therefore, the TaqMan assay should be used as a substitute for EM analysis for retrovirus-like particle quantification in CHO cell-based production system.