Stark Quantitative HBV Molecular Diagnostic Kit

The Stark Quantitative HBV Molecular Diagnostic Kit is intended for in vitro diagnostic use by healthcare professionals. Purposefully crafted for the qualitative detection and quantitative determination of Hepatitis B Virus (HBV) DNA in blood samples, the results derived from this test are intended for clinical interpretation. They serve to aid healthcare professionals in diagnosing HBV infections, monitoring disease progression, and informing treatment decisions. Meticulous adherence to the provided instructions is imperative to ensure the attainment of accurate and dependable results.

The Stark Quantitative HBV Molecular Diagnostic Kit offers a convenient and ready-to-use system for detecting Hepatitis B Virus DNA utilizing polymerase chain reaction (PCR) along with primers and specific fluorescent probes.

Sample collection

Storage and sample transportation

• Transport samples following specific precautionary procedures for pathogens, ensuring transportation does not exceed six hours.
• All samples must be transported at temperatures ranging from 2°C to 8°C, while plasma samples should be kept at -20°C.
• Whole blood should undergo separation into plasma and cellular components via centrifugation at 1200-1600 rpm for 20 minutes. Transfer the extracted plasma into sterile Eppendorf tubes.
• Avoid freezing blood samples, as routine freezing or prolonged storage can potentially reduce the assay’s sensitivity.
• After extraction, isolated Hepatitis B Virus encapsulated DNA remains stable for up to 14 days if stored at +4°C, for 12 weeks if stored at -20°C, and up to one year when stored at -70°C.

Before use

Remove each component from the kit and place them on the benchtop. Allow the reagents to equilibrate to room temperature. Subsequently, briefly vortex each tube in preparation for later use.

Kit Contents Introduction

For mass screenings, swift and precise detection of HBV is paramount. Real-Time PCR HBV kits, boasting high sensitivity, fulfill these requirements adeptly. Apart from the specific amplification of the HBV genome, this method incorporates oligonucleotides for the direct detection of the internal control (IC). The IC should be manually added at the onset of the nucleic acid purification procedure. Probes specific to Hepatitis B Virus DNA are labeled with the fluorophore FAM™ (green), while the IC is labeled with a fluorophore detectable in the VIC™ (yellow) channel.

QR-ROMAX and Pro HBV Mix solutions

These solutions encompass all essential components, including PCR buffer, DNA polymerase enzyme, magnesium salt, primers, and probes, facilitating the PCR-mediated amplification and target detection of HBV-specific DNA and the internal control (IC) in a single reaction setup.

Quantification Standards (QS)

The Quantification Standards (QS) comprise standard concentrations of HBV-specific DNA (refer to Table 1). These standards are calibrated against the International Standard for HBV DNA for Nucleic Acid Amplification Techniques and adhere to the Clinical Laboratory Standards Institute guidelines. The Quantification Standards serve the purpose of validating the functionality of the HBV DNA-specific amplification and detection system. Additionally, they are utilized to generate a standard curve, facilitating the quantification of HBV-specific DNA in a sample.

Table 1: Quantification Standards

• NTC: No Template Control.
• NTC: Contains no HBV-specific DNA but includes the Internal Control template.
• The NTC serves as a negative control for the HBV DNA-specific Real-Time PCR and signifies potential contamination of QR-ROMAX and Pro HBV mix.

Recommended Starting Material

DNA Sample Requirement

Prior to commencing, add a 2-5cc blood sample into a tube containing EDTA. Following plasma isolation and DNA extraction, employ 10μl of the entire prepared sample in Real-Time PCR.

Sample Storage and Preparation

• The Stark Quantitative HBV Molecular Diagnostic Kit is intended for use with human EDTA plasma samples. Other sample materials have not been validated.
• Blood should be collected using commercially available standard blood collection systems (e.g., Sarstedt, Becton Dickinson, Greiner, or equivalent).
• Blood samples should be kept cooled at temperatures ranging from 2°C to 8°C.
• To generate EDTA plasma, whole blood should be centrifuged according to the instructions provided by the manufacturer of the collection system within 24 hours after collection.
• EDTA plasma should not be stored for more than two days at room temperature (20°C to 25°C), five days at 2°C to 8°C, or two months at -25°C to -15°C before use.
• Always handle samples as infectious and (bio-)hazardous, adhering to safe laboratory procedures. Promptly use an appropriate disinfectant for sample material spills and treat contaminated materials as biohazardous.
• Frozen storage of samples does not compromise the performance of the kit. Ensure that frozen samples are completely thawed and properly mixed before use.
• Exercise caution regarding the health risks associated with positive samples, following necessary precautionary procedures at all stages, from collection and transportation to kit application.
• Note that EDTA is the most suitable anticoagulative buffer for the Stark Quantitative HBV Molecular Diagnostic Kit.
• The use of other anticoagulants may not guarantee proper function and results.

Caution: Samples collected in tubes containing heparin as an anticoagulant should not be used.

Before start

• Prior to first use, ensure the intactness and completeness of the kit contents and reagents.
• Avoid using samples other than human plasma to prevent incorrect in vitro diagnostic (IVD) examination results.
• Misuse of the reagents may result in contamination and invalid results.
• Utilize RNAse/DNAse free pipette tips with filters for sampling.

Buffer preparation

Refer to Table 3 for the required information to prepare the buffer.

Master Mix Preparation

Prepare the Master Mix according to the information provided in Table 4 and Table 5. Ensure to prepare the Master Mix for single-use only. Avoid adding QR-ROMAX to Pro HBV Mix if testing is not required. If using the internal control, refer to the relevant information in the handbook.

Table2: Reagents preparation per one single reaction (DNA isolation efficiency and PCR inhibition are controlled by adding internal control in the purification stage)

Table 3: Required volumes for standard tubes

Table 4: Required volumes for every single test tube

Table 5: Required volumes for negative control tubes

Notice: Pay attention to using the NTC tube in each run.

*Sample is changed with water in NTC tube, controlling contamination in reaction.

Pathogenesis

The Hepatitis B Virus (HBV) is responsible for the disease hepatitis B and possesses unique characteristics among human viral pathogens. It is a DNA virus that replicates via an RNA intermediate, placing it within the category of reverse transcribing DNA and RNA viruses. HBV belongs to the Hepadnaviridae family of viruses, which comprises genotypes A-H. The genomic structure of HBV is compact yet complex, capable of encoding seven distinct proteins within its 3.2 kb genome. These proteins include the polymerase protein (Pol gene); core antigen (HBsAg) and e antigen (HBeAg); large, medium, and small surface-antigen proteins (S gene); and the X protein (X gene).

Transmission of HBV occurs primarily through blood or other body fluids and can survive outside the body for up to seven days. Common transmission routes include perinatal mother-to-infant transmission or horizontal transmission among children under 5 years old. Sources of infection may also include medical instruments used in surgery, tattooing needles, or razors contaminated with blood. The virus typically manifests 30 to 60 days after infection and can persist in the body, potentially leading to the development of chronic hepatitis B.

Symptoms of hepatitis B infection can vary from jaundice (yellowing of the skin and eyes) and dark urine to extreme fatigue, nausea, vomiting, and abdominal pain. Symptoms may persist for several weeks, although carriers of the virus can remain asymptomatic. The most severe outcomes include acute or chronic hepatitis, which may progress to liver cirrhosis or hepatocellular carcinoma (HCC). Currently, there is no cure for hepatitis B, but medications are available for symptom management and slowing the progression of cirrhosis.

Despite vaccination efforts, HBV infections remain prevalent worldwide, with approximately 240 million people suffering from chronic HBV infection and 887,000 HBV-related deaths annually (numbers increasing since 2015). The highest prevalence of Hepatitis B Virus is observed in the Western Pacific and Africa, where 6.2% and 6.1% of the adult population are infected, respectively. Infections are also prevalent in WHO-specified regions such as the Eastern Mediterranean, South-East Asia, Europe, and the Americas. Hence, there is a critical need for viral hepatitis B testing as an essential component of prevention and treatment efforts.

Workstation Preparation

All work areas, samplers, centrifuges, and related equipment must maintain sterility throughout the testing process. In case of nucleic acid contaminations, employ decontaminants such as Sodium hypochlorite 10%, ethanol 70%, and RNZO to ensure thorough decontamination.

Figure 1: Addition of internal control during DNA amplification in PCR. In this case, Master mix involves Pro HBV Mix and QR-Romax,4×. For more information, refer Master mix preparation.

Figure2: Addition of internal control to Master mix. Notice that there is not any addition of internal control during the purification stage. In this case, Master mix involves Pro HBV Mix and QR-Romax,4×. For more information, refer Master mix preparation.

Table 6: PCR program for HBV

Process

• Switch on the ABI Step One/Step One Plus instrument.
• Turn on the computer, and launch the ABI Step One/Step One Plus software.
• Begin the PCR setup according to the PCR Run Program outlined in Table 6.
• Ensure acquisition is obtained in both the green channel (HBV target detection channel) and the yellow channel (IC target detection channel). Refer to the ABI Step One/Step One Plus instruction manual for detailed guidance.
• Upon launching the ABI Step One/Step One Plus software, open a new experiment window by selecting “New Experiment,” and fill in the required information and select the intended options as depicted in Figure 1.
• Depending on the instrument brand, choose the appropriate options:

• For StepOnePlus instrument (96 wells) or StepOne instrument (48 wells)
• Select “Quantitation standard curve” based on the Stark Quantitative HBV Molecular Diagnostic Kit.
• Choose “TaqMan reagents.”
• Select “Standard” run mode, which takes approximately 2 hours to complete a run.

• In the next step, click on “plate setup” and define the first and second targets as FAM and VIC/HE, respectively, and identify their colors as green and yellow (refer to Figure 2).
• Click on “Assign target and sample” and enter both the sample and IC names based on the order in which the wells are filled. Set the Passive reference state to ROX.
• For identifying standards, in the “Assign target and sample” dialogue, define each well for each standard and its concentration in the “Define and set up standards” section (refer to Figure 4).
• In the “Run method” dialogue, optimize the PCR program based on the aforementioned thermal profile in the kit’s protocol. Click on “Add stage/step” to add more steps if needed. Click on “Run” and then “Save” to start the reaction.

The Stark Genotyping HPV Molecular Diagnostic Kit undergoes rigorous testing through predetermined experiments on a lot-to-lot basis to guarantee consistent product quality. Accessible results of these experiments can be obtained online by referencing the REF and Lot numbers at www.amorph.tech.

The Quantification Standards provided in the Stark Quantitative HBV Molecular Diagnostic Kit utilize a standard panel comprising identified concentrations of HBV DNA. When preparing both sample and standard concentrations, add 10µl of isolated DNA to the 15µl Master Mix. The Quantification Standards serve to generate the standard curve, enabling the quantification of HBV-specific DNA concentration in the sample.

Enter the qualification standard in the ABI-specific software in IU/ml. Follow the provided formula to convert IU/µl, as supplied by the kit contents.

If the volume of whole plasma were 200µl and elution 50µl, the first standard would be 2.5×10⁷ IU/ml entered in ABIe-specific software.

• All reagents are exclusively intended for in-vitro diagnostics.
• The product is to be used by personnel specially instructed and trained in in-vitro diagnostics.
• Strict adherence to the user manual is necessary for optimal PCR results.
• Attention should be given to the expiration dates printed on the box and labels of all components. Do not use expired components.

Storage

All components of the Stark Quantitative HBV Molecular Diagnostic Kit are pre-prepared and ready for immediate use upon arrival. Upon receipt, it is recommended to store all reagents at temperatures ranging from -15°C to -30°C. These conditions ensure stability and maintain the integrity of the components until the expiration date indicated on the label.

Warning and Precautions

• This kit is intended for in vitro diagnostic use only.
• Adhere diligently to laboratory safety protocols.
• Familiarize yourself thoroughly with the guidelines before usage.
• Refrain from eating, drinking, smoking, chewing gum, applying cosmetics, or taking medicine in laboratories where hazardous materials and human samples are handled.
• Treat all patient samples and positive controls as potentially infectious.
• Use the Stark Genotyping HPV Molecular Diagnostic Kit under the supervision of a physician for emergency and in vitro diagnostic purposes.
• Ensure all procedure steps, including sampling, storage, shipping, and laboratory tests, comply with biosafety and molecular laboratory management standards.
• Equip clinical laboratories with instruments and operators in accordance with the regulations of the Ministry of Health.
• Any alteration or replacement of kit contents may affect functionality and contravene product licensing.
• Utilize sterile and DNase-RNase-free pipette tips and microtubes to prevent contamination. Change filter pipette tips after each substance or sample addition.
• Dispose of waste following biosafety guidelines. Regularly sanitize desks and laboratory instruments with 70% Ethanol or 10% Sodium Hypochlorite.
• Shield Pro Mixes from sunlight exposure.
• Promptly clean and disinfect specimen spills using suitable disinfectants adhering to national and local regulations.
• Dispose of all specimens, reagents, and potentially contaminated materials following national and local regulations.
• The hazard and precautionary statements provided apply to the components of the Stark Genotyping HPV Molecular Diagnostic Kit.