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PubMed2004

Establishment and assessment of two methods for quantitative detection of serum duck hepatitis B virus DNA.

AIM: To establish and assess the methods for quantitative detection of serum duck hepatitis B virus (DHBV) DNA by quantitative membrane hybridization using DHBV DNA probe labeled directly with alkaline phosphatase and fluorescence quantitative PCR (qPCR). METHODS: Probes of DHBV DNA labeled directly with alkaline phosphatase and chemiluminescent substrate CDP-star were used in this assay. DHBV DNA was detected by autoradiography, and then scanned by DNA dot-blot. In addition, three primers derived from DHBV DNA S gene were designed. Semi-nested primer was labeled by AmpliSensor. Standard curve of the positive standards of DHBV DNA was established after asymmetric preamplification, semi-nested amplification and on-line detection. Results from 100 samples detected separately by alkaline phosphatase direct-labeled DHBV DNA probe with dot-blot hybridization and digoxigenin-labeled DHBV DNA probe hybridization. Seventy samples of duck serum were tested by fluorescent qPCR and digoxigenin-labeled DHBV DNA probe in dot-blot hybridization assay and the correlation of results was analysed. RESULTS: Sensitivity of alkaline phosphatase direct-labeled DHBV DNA probe was 10 pg. The coincidence was 100% compared with digoxigenin-labeled DHBV DNA probe assay. After 30 cycles, amplification products showed two bands of about 180 bp and 70 bp by 20 g/L agarose gel electrophoresis. Concentration of amplification products was in direct proportion to the initial concentration of positive standards. The detection index was in direct proportion to the quantity of amplification products accumulated in the current cycle. The initial concentration of positive standards was in inverse proportion to the number of cycles needed for enough quantities of amplification products. Correlation coefficient of the results was (0.97, P<0.01) between fluorescent qPCR and dot-blot hybridization. CONCLUSION: Alkaline phosphatase direct-labeled DHBV DNA probe in dot-blot hybridization and fluorescent qPCR can be used as valuable means to quantify DHBV DNA in serum.

Alkaline Phosphatase↗
PubMed2004

Optimization of in situ hybridization assay using non-radioactive DNA probes for the detection of canine herpesvirus (CHV) in paraffin-embedded sections.

Two non-radioactive probes using digoxigenin or biotin were developed for detecting canine herpesvirus (CHV) and compared for their sensitivities by in situ hybridization (ISH) in formalin fixed, paraffin embedded sections, which has been used routinely in veterinary fields. Sections of the CHV-infected cell preparation were subjected to several different ISH protocols using digoxigenin- or biotin-labeled probe respectively. Results were compared for the hybridization and background signal intensities. The best result was obtained by the optimized ISH protocol using digoxigenin-labeled probe for detection of CHV DNA. The optimized ISH assay, which developed in this study, may be a valid tool for the study of pathogenesis and diagnosis of CHV infection.

Animals↗
PubMed2006

Localization of cannabinoid CB1 receptor mRNA using ribonucleotide probes: methods for double- and single-label in situ hybridization.

This chapter presents a reliable, detailed method for performing double-label in situ hybridization (ISH) that has been validated for use in studies identifying the co-localization of cannabinoid CB1 receptor mRNA with other distinct species of mRNAs. This method permits simultaneous detection of two different species of mRNA within the same tissue section. Double-label ISH may be accomplished by hybridizing tissue sections with a combination of radiolabeled and digoxigenin-labeled RNA probes that are complementary to their target mRNAs. Single-label ISH may be accomplished by following the procedures described for use with radioisotopic probes (here [35S]-labeled) only. Silver grains derived from conventional emulsion autoradiography are used to detect the radiolabeled cRNA probe. An alkaline phosphatase-dependent chromogen reaction product is used to detect the nonisotopic (here, digoxigenin-labeled) cRNA probe. Necessary controls that are required to document the specificity of the labeling of the digoxigenin and radiolabeled probes are described. The methods detailed herein may be employed to detect even low levels of a target mRNA. These methods may be utilized to study co-localization and coregulation of expression of a particular gene within identified neurons in multiple systems.

Animals↗
PubMed1983

Cross reactivity of the EMIT digoxin assay with digoxin metabolites, and validation of the method for measurement of urinary digoxin.

In evaluating the EMIT (Syva Co.) digoxin assay, we found no cross reactivity between dihydrodigoxin, the major digoxin metabolite, and the EMIT digoxin antibody from two different lots. However, the antibody does cross react, essentially completely, with the digoxin hydrolysis metabolites digoxigenin, digoxigenin mono-digitoxide, and digoxigenin bis-digitoxide. The EMIT method can be used to measure digoxin in urine diluted at least 50- to 100-fold with digoxin-free human plasma; the inter-assay coefficient of variation of this assay is 6%. In addition, we validated the manual EMIT serum digoxin assay, using external ("TRI-rac") quality controls.

Cross Reactions↗
PubMed1994

Digoxin immunoassay with cross-reactivity of digoxin metabolites proportional to their biological activity.

Our objective was to identify commercially available digoxin immunoassays whose cross-reactivity with digoxin metabolites paralleled the pharmacological activity of the metabolites. We measured the immunoreactivity of digoxigenin bis- and monodigitoxosides, digoxigenin, and dihydrodigoxin in four immunoassays and compared the immunoactivities with pharmacological activities from studies involving whole-animal and receptor (Na,K-ATPase)-based assays. Correlation coefficients for comparisons of immunoassay reactivity and human heart receptor reactivities were: ACS, 0.96; TDx, 0.60; Stratus, 0.57; and Magic, 0.42. Comparison with other biological assays showed a similar trend. The major difference in metabolite cross-reactivities among the immunoassays was that of digoxigenin (ACS, 0.7%; TDx, 103%; Stratus, 108%; Magic, 153%), which has approximately 10% bioactivity relative to digoxin. Measured recovery of mixtures of digoxin and metabolites confirmed these findings. We conclude that the monoclonal antibody in the ACS digoxin assay closely mimics Na,K-ATPase in detecting digoxin and its metabolites. This finding provides a basis for developing therapeutic drug monitoring immunoassays capable of approximating the true pharmacological activity of a mixture of drug metabolites.

Animals↗
PubMed1996

Multiparametric cell-cycle analysis of peripheral blood-activated lymphocyte subsets using staining based on the TdT method for incorporated BrdUrd.

This study describes a new method for the simultaneous assessment of the distribution of a cell population in the G0/G1, S, and G2/M cell-cycle phases by using multiparameter flow cytometry and single staining based on BrdUrd incorporation. Both the K562 cell line and PHA-stimulated peripheral blood lymphocytes (PBL) were analyzed. Cells were cultured in the presence of BrdUrd for 30 min prior to cell harvesting. Once collected, cells were exposed to ultraviolet light for 5 min and then fixed immediately in 70% ethanol (-20 degrees C) for at least 30 min. Once fixed, the cells were placed for 30 min at 37 degrees C in the presence of terminal deoxynucleotidyl transferase (TdT) and dUTP labeled with digoxigenin; they were then stained with FITC-labeled anti-digoxigenin. Our results show that G0/G1, s, and G2/M cell populations can be clearly discriminated according to FITC fluorescence and light-scatter parameters. In this way, S-phase cells can be identified by their FITC staining. From the cells which were negative for anti-digoxigenin-FITC antibody, two clear populations could be resolved in a forward scatter, side scatter, and fluorescence pulse-width three-dimensional plot; the values obtained for G0/G1 cells were lower than those obtained for G2/M cells in all three parameters. Multiparameter analysis of PBL stained for two surface antigens (CD3 and CD8) and for BrdUrd by direct or indirect TdT method permitted cell-cycle analysis of different subpopulations, including CD3+/CD8+, CD3+/CD8-, CD3-/CD8+, and CD3-/CD8-.

Bromodeoxyuridine↗
PubMed2000

Localization of cannabinoid CB(1) receptor mRNA in neuronal subpopulations of rat striatum: a double-label in situ hybridization study.

Double-label in situ hybridization was used to identify the phenotypes of striatal neurons that express mRNA for cannabinoid CB(1) receptors. Simultaneous detection of multiple mRNAs was performed by combining a (35)S-labeled ribonucleotide probe for CB(1) mRNA with digoxigenin-labeled riboprobes for striatal projection neurons (preprotachykinin A, prodynorphin, and preproenkephalin mRNAs) and interneurons (vesicular acetylcholine transporter (VAChT), choline acetyltransferase (ChAT), somatostatin, and glutamic acid decarboxylase (Mr 67,000; GAD67) mRNAs). To ascertain whether CB(1) mRNA was a marker for striatal efferents, digoxigenin-labeled probes for mRNA markers of both striatonigral (prodynorphin or preprotachykinin A mRNAs), and striatopallidal (proenkephalin mRNAs) projection neurons were combined with the (35)S-labeled probe for CB(1). A mediolateral gradient in CB(1) mRNA expression was observed at rostral and mid-striatal levels; in the same coronal sections the number of silver grains per cell ranged from below the threshold of detectability at the medial and ventral poles to saturation at the dorsolateral boundary bordered by the corpus callosum. At the caudal level examined, CB(1) mRNA was denser in the ventral sector relative to the dorsal sector. Virtually all neurons expressing mRNA markers for striatal projection neurons colocalized CB(1) mRNA. Combining a (35)S-labeled riboprobe for CB(1) with digoxigenin-labeled riboprobes for both preproenkephalin and prodynorphin confirmed localization of CB(1) mRNA to striatonigral and striatopallidal neurons expressing prodynorphin and preproenkephalin mRNAs, respectively. However, CB(1) mRNA-positive cells that failed to coexpress the other markers were also apparent. CB(1) mRNA was localized to putative GABAergic interneurons that express high levels of GAD67 mRNA. These interneurons enable functional interactions between the direct and indirect striatal output pathways. By contrast, aspiny interneurons that express preprosomatostatin mRNA and cholinergic interneurons that coexpress ChAT and VAChT mRNAs were CB(1) mRNA-negative. The present data provide direct evidence that cannabinoid receptors are synthesized in striatonigral neurons that contain dynorphin and substance P and striatopallidal neurons that contain enkephalin. By contrast, local circuit neurons in striatum that contain somatostatin or acetylcholine do not synthesize cannabinoid receptors. Published 2000 Wiley-Liss, Inc.

Animals↗
PubMed1976

Studies on digitalis. V. The influence of impaired renal function, hemodialysis, and drug interaction on serum protein binding of digitoxin and digoxin.

The aim of the present investigation is to study digitoxin and digoxin protein binding in patients with normal renal and hepatic function, in patients with uremia, and in patients under treatment with hemodialysis for renal failure. The binding of digitoxin and cardioactive metabolites to serum proteins was studied using equilibrium dialysis (an in vitro chemical assay) alone and in combination with a modified 86Rb method. The following values for protein binding were found: DT-3 (digitoxin), 95.7%; DT-2 (digitoxigenin-bis-digitoxoside), 96.5%; DT-1 (digitoxigenin-mono-digitoxoside), 98.7%; DT-0 (digitoxigenin), 92.7%; DG-3 (digoxin), 21.2%; DG-2 (digoxigenin-bis-digitoxoside), 16.3%; DG-1 (digoxigenin-mono-digitoxoside), 18.5%; and DG-0 (digoxigenin), 13.3%. In vitro addition of procainamide, phenytoin, heparin, and rifampicillin did not influence the in vitro binding of digitoxin. Protein binding of digitoxin showed small individual variations in patients with normal renal and hepatic function. Uremia per se did not influence the in vitro binding of digitoxin. There were marked changes in digitoxin and digoxin protein binding during an 8-hr hemodialysis, digitoxin binding decreasing from 97.1% to 93.7% (p less than 0.0025) and digoxin binding from 23.5% to 15.4% (p less than 0.05). In the uremic patient the metabolic pattern of digitoxin tended toward a decrease in protein-bound metabolites.

Blood Proteins↗
PubMed1979

Biotransformation and elimination of digoxin with normal and minimal renal function.

Six subjects with normal renal function (NRF) and 6 patients with minimal renal function (MRF) on 3 times weekly hemodialysis received 150 muCi3H-digoxin-12 alpha orally. Serial urine collections were made for five days or more. Digoxin and metabolites were separated using diethylaminoethyl Sephadex LH-20 column chromatography. Mean cumulative percentages of the ingested radioactivity excreted over five days in NRF and MRF groups were: digoin, 54.5% and 14.7%; bis-digitoxoside of digoxigenin, 2.0% and 0.59%; mono-digitoxoside, 0.8% and 0.19%; digoxigenin, 0.25% and 0.03%; and dihydrodigoxin, 0.3% and 0.03%. Half-lives based on the mean rates of disappearance from urine comparing NRF and MRF groups were: for digoxin 40 hr and 120 hr; for bis-digitoxoside, 11.5 hr and 46 hr; for mono-digitoxoside, 8.5 hr and 12 hr; for digoxigenin, 2 hr and 7.5 hr; and for dihydrodigoxin, 1.2 hr and 7.0 hr. Considering the relationships of the five-day cumulative excretion and half-lives of digoxin and metabolites in the NRF and MRF groups, it appears unlikely that there is a major alteration in the biotransformation of digoxin in advanced renal failure when there appears to be a shift from renal to slower biliary excretion.

Adult↗
PubMed1998

Optimization and automation of fluorescence-based DNA hybridization for high-throughput clone mapping.

Large-scale hybridization-based genome mapping projects, such as the production of sequence-ready physical clone maps, call for robust and cheap DNA labeling techniques that are amenable to automation. We routinely use a high-throughput protocol based on fluorescence detection. DNA probes are labeled via polymerase chain reaction (PCR) amplification with primers that are digoxigenin-modified at their 5' ends. Alternatively, digoxigenin-labeled dUTP is incorporated in a random hexamer priming reaction. Hybridization takes place in small volumes by sandwiching the probe between filters and plastic sheets. A fluorescence signal is produced by the activity of alkaline phosphatase attached to an anti-digoxigenin antibody upon the addition of AttoPhos substrate. Signals are directly detected with a charge-coupled device (CCD) camera and scored by an image data analysis system. DNA filters can be reused at least 40 times without loss of data quality. Significant advantages compared to radioactive techniques are the reduced health risk, enabling highly parallel processing; the production of spot signals uniform in size and intensity, which is essential for efficient image analysis; and a cost reduction of about 70%.

Alkaline Phosphatase↗
PubMed1982

Influence of 16 beta formylation on Na, K-ATPase inhibition by cardiac glycosides.

The inhibitory effect of formylated cardiac steroids (gitaloxin and its derivatives) on guinea-pig heart Na, K-ATPase was compared to that of other cardiac steroids with various hydroxy substituents. The decreasing order of potency of aglycones at equilibrium was as follows: gitaloxigenin greater than digitoxigenin greater than ouabagenin greater than digoxigenin greater than gitoxigenin greater than diginatigenin. This sequence was different to the sequence of drugs hydrophobic character. The compounds with hydroxy groups in the vicinity of the lactone ring (gitoxigenin, diginatigenin) were less potent than the hydrophilic compound ouabagenin. We propose that intramolecular bounding between 16 beta-OH and the lactone ring contributes to the relatively low potency of gitoxigenin and diginatigenin. The formylation of 16 beta-OH increased the potency of gitoxigenin by a factor of 41. The formylated compound (gitaloxigenin) was 5-fold more potent than digitoxigenin. The 3 beta-glycosylation of digoxigenin lead to pseudo-irreversible inhibitors of Na, K-ATPase. The half-time to achieve the equilibrium (for 5 mumol/l) was equal to 54 s, 90 s and 108 s respectively for digoxigenin monodigitoxoside, digoxin and desacetyllanatoside C. However, at equilibrium the three glycosides were equipotent, suggesting the existence of steric effects at the sugar site of the receptor. The sequence of potency observed for monodigitoxosides, monodigitalosides and tridigitoxosides after 60 min incubation was similar to that observed for the corresponding aglycones. These results suggest that the strongly negative inductive group 16 beta-OCHO is tightly bound to Na, K-ATPase, possibly to the same receptor site than that which is thought forming hydrogen and ionic bonds with the lactone ring. They show that the high toxicity of gitaloxin in guinea-pig heart is likely due to its high potency as Na, K-ATPase inhibitor.

Animals↗
PubMed1998

Improved mRNA in situ hybridization on formaldehyde-fixed and paraffin-embedded tissue using signal amplification with different haptenized tyramides.

We report an optimized in situ hybridization (ISH) protocol with a rapid signal amplification procedure based on catalyzed reporter deposition (CARD) to increase the sensitivity of non-isotopic mRNA ISH on formaldehyde-fixed and paraffin-embedded tissue. The CARD method is based on the deposition of haptenized tyramide molecules in the vicinity of hybridized probes catalyzed by horseradish peroxidase. Commercially available and newly synthesized haptenized tyramides, including digoxigenin-, biotin-, di- and trinitrophenyl- as well as fluorescein-tyramide, were compared. The haptenized tyramides were visualized using peroxidase conjugated anti-hapten antibodies followed by the diaminobenzidine reaction. As a test system, we applied digoxigenin-labeled oligonucleotides to detect insulin and vasoactive intestinal polypeptide mRNA in pancreatic endocrine tumors and liver metastases. Our results indicate that specificity, sensitivity, and applicability of oligonucleotide mRNA ISH can be significantly improved by using chemically digoxigenin-labeled oligonucleotide probes and signal amplification by CARD. Furthermore, all tested tyramides provided approximately equal amplification efficiency. In conclusion, CARD signal amplification should further promote mRNA ISH studies on paraffin-embedded tissues and allow for multiple-target nucleic acid detection in situ.

Dinitrophenols↗
PubMed1992

Chemiluminescent microtiter method for detecting PCR amplified HIV-1 DNA.

A rapid and sensitive method for detecting HIV-1 DNA sequences amplified by polymerase chain reaction (PCR) is described. The method uses solution phase hybridization for rapid and efficient annealing between digoxigenin-labeled targets and biotinylated capture probes. Hybrids containing biotin are captured onto streptavidin coated microwells and all other PCR components are washed away, including spurious amplification products. The presence of the digoxigenin-labeled amplified HIV target is then detected by anti-digoxigenin-alkaline phosphatase conjugates using the chemiluminescent substrate PPD. This approach maintains high specificity by nucleic acid dependent capture, and high sensitivity by efficient solution hybridization. The method is rapid (2 hours), and capable of detecting 10 HIV targets.

Alkaline Phosphatase↗
PubMed1996

Rapid detection of enterovirus in clinical specimens using PCR and microwell capture hybridization assay.

A rapid detection method of enteroviral RNA in clinical samples using PCR and a microwell capture hybridization assay is described. PCR products were labelled directly by digoxigenin-dUTP during the amplification step. The labelled amplicons were hybridized with a biotinylated oligo-probe and captured on commercially available test microwells coated with streptavidin. The hybridized amplicons labelled with digoxigenin were detected using anti-digoxigenin Fab fragments conjugated to peroxidase and colorimetric reaction automatically measured. This method detected as few as 0.01 PFU/100 microl of biological sample with a result obtained within 8 h. Using this method, we were able to detect enteroviral RNA in 23 of 35 clinical specimens from 16 of 17 patients with suspected acute or chronic enteroviral infection. The samples included cerebrospinal fluid, broncho-pulmonary lavage, pericardial effusion, throat swabs, stools, sera, muscular and myocardial biopsies. In contrast, virus was isolated in cell culture in only 8 of 28 clinical specimens from 6 of the 17 patients. This easy-to-perform assay has useful potential in the rapid detection of enterovirus in acute or chronic infection. This methodology could be used for a rapid qualitative detection of other RNA viruses.

Enterovirus↗
PubMed1993

Co-expression of four muscarinic receptor genes by the intrinsic neurons of the rat and guinea-pig heart.

Expression of the messenger RNAs encoding the five different muscarinic acetylcholine receptor subtypes was examined in intracardiac neurons from the rat and guinea-pig heart by in situ hybridization techniques. Newborn guinea-pig intracardiac neurons were studied in dissociated cell culture preparations employing both 35S- and digoxigenin-labelled oligonucleotide probes specific for the m1, m2, m3, m4 or m5 muscarinic receptor messenger RNAs. When 35S-tailed oligonucleotides were used, all intracardiac neurons in culture were found to express m1, m2, m3 and m4, but not m5 messenger RNAs. However after hybridization with digoxigenin-tailed probes, only m1 and m2 transcripts were detected. This may reflect differences in the sensitivity of the two techniques. Further to these experiments, intracardiac ganglia in sections of adult rat heart were studied employing m1-, m2-, m3- or m4-specific, 35S-labelled oligonucleotides, and again, all intracardiac neurons expressed messenger RNA for each of these four muscarinic receptor subtypes. Atrial myocytes in culture were only labelled by [35S]- and digoxigenin-tailed m2 oligonucleotides. No other heart cell type seen expressed messenger RNA for any of the muscarinic receptors. The expression of four different muscarinic receptor transcripts by intrinsic neurons of the heart provides the molecular basis for the diverse muscarinic actions observed in these and other autonomic ganglia.

Animals↗
PubMed1995

Specific and sensitive determination of digoxin and metabolites in human serum by high performance liquid chromatography with cyclodextrin solid-phase extraction and precolumn fluorescence derivatization.

A precolumn fluorescence derivatization high performance liquid chromatographic method has been developed for the simultaneous determination of digoxin and its metabolites digoxigenin bisdigitoxoside, digoxigenin monodigitoxoside digoxigenin, and dihydrodigoxin (20-R and 20-S epimers) in human serum. Digoxin and its metabolites were extracted from serum samples (containing digitoxin as internal standard) with a cyclodextrin solid-phase extraction (SPE) column. Fluorescent derivatives were formed by reaction of the analytes with 1-naphthoyl chloride in the presence of 4-dimethylaminopyridine under a nitrogen atmosphere in a glove box with controlled relative humidity (26% r.h. or less). The derivatives were isolated using cyclodextrin and C1 SPE columns sequentially, and determined by HPLC using silica column separation and fluorescence detection. Calibration curves were linear over the concentration range from 0.25 to 4.0 ng ml-1. Recoveries of digoxin and its metabolites from serum ranged from 62 to 86%, and coefficients of variation from repetitive analyses ranged from 6.9 to 20.9% and from 5.8 to 12.2% at 0.5 ng ml-1 and 2.0 ng ml-1, respectively. This method has been shown capable of specifically determining digoxin and its major metabolites in serum, and has been successfully used in the determination of digoxin and its metabolites in serum samples collected from patients undergoing digoxin therapy. This method thus permits the investigation of digoxin metabolism and pharmacokinetics after the administration of commercial dosage forms.

Calibration↗
PubMed2002

A method for the detection and screening of catalytic anti-DNA antibodies.

We have developed a microtiter plate assay for the detection and screening of anti-DNA hydrolytic antibodies. The affinity-linked oligonucleotide nuclease assay (ALONA) makes use of substrates with a digoxigenin on the 5'-end of the 3'-biotinylated DNA strands. The substrate binds specifically to the wells of streptavidin-coated microtiter plates where the reaction takes place. Uncleaved substrate retains the digoxigenin label, which is then detected with an enzyme-labeled anti-digoxigenin antibody. We first assessed the efficiency of this assay by measuring S1 nuclease and DNase I activities and the inhibitory effect of EDTA on the reaction. The ALONA procedure was then successfully applied to the screening of a high number of hybridoma clones derived from nonimmunized (NZB x NZW)F1 mice with spontaneous lupus erythematosus. We detected three potential catalytic antibodies and investigated their substrate specificity. Overall, our findings demonstrate the value of the ALONA method for high throughput screening of potential nucleases and catalytic antibodies. Although this assay was designed for the selection of catalysts active in DNA hydrolysis, it can be adapted to detect most types of substrate cleavage reaction.

Animals↗
PubMed2003

Multiplex RT-PCR-ELISA compared with bioassay for the detection of four apple viruses.

A sensitive and reliable multiplex RT-PCR-ELISA technique for the detection of Apple chlorotic leaf spot virus, Apple stem pitting virus, Apple mosaic virus and Apple stem grooving virus was developed. This technique is compared with the method used commonly for indexing by woody indicators, which is time consuming and expensive. For the RT-PCR-ELISA technique, the amplified products were labeled with digoxigenin during the RT-PCR by incorporation of a digoxigenin labeled primer. After hybridization of the PCR products to specific capture oligonucleotides, which were bound covalently to the surface of NucleoLink strips, anti-digoxigenin antibodies were used for detection. More than 100 samples were tested in parallel by indexing and multiplex-RT-PCR-ELISA. All infections detected by woody indicators were also detected by multiplex RT-PCR-ELISA. Furthermore, additional infections were only found by multiplex RT-PCR-ELISA. The colourimetric detection of multiplex-RT-PCR products was at least as sensitive and sometimes slightly more sensitive than detection by gel electrophoresis. The results show that this molecular technique is more reliable for the detection of the above mentioned apple viruses than indexing by woody indicators, thereby helping to reduce cost and time during the certification of plant material.

Biological Assay↗