Stark Genotyping HPV Molecular Diagnostic Kit

The HPV Genotyping Molecular Diagnostic Kit (Real-Time PCR) is intended for in vitro diagnostic use. This kit is designed for the qualitative detection and genotyping of human papillomavirus (HPV) DNA in cervical or vaginal specimens. Results obtained are for professional use only by trained and validated laboratory personnel. This kit aids in identifying HPV infections, assisting healthcare professionals in making informed decisions regarding patient management and treatment strategies. Careful adherence to provided instructions is crucial to ensure accurate and reliable results.

The Stark Genotyping HPV Molecular Diagnostic Kit technology is an in vitro nucleic acid TaqMan assay employing signal amplification through polymerase chain reaction and fluorescent probes (ROX/Texas Red, Yakima Yellow, and FAM). It is utilized for the genotyping detection of 14 high-risk and two low-risk types of HPV DNA in cervical or vaginal specimens. The high-risk HPV types detected include 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, along with low-risk types 6 and 11. This diagnostic test kit is applicable in human cervical screening, cytology samples, urine, and paraffin-embedded tissue analysis. Utilizing the polymerase chain reaction (PCR), the kit is configured with three-Channels Multiplex Real-Time PCR instruments.

Materials Required (but Not Provided)

• DNase-RNase-free microtubes (1.5ml)
• PCR microtube 0.1ml or 0.2ml strip
• Various models of pipettes and pipette tips (10µl, 100µl, and 1000µl filter pipette tips)
• Surface sanitizing solution
• Disposable Powder-Free gloves and surgical gown
• Three-Channels Multiplex Real-Time PCR Instrument (with green, yellow, and orange channels)
• Vortex
• Cool box

Procedures

The Stark Genotyping HPV Molecular Diagnostic Kit employs a polymerase chain reaction of Real-Time PCR for its testing procedure. Specifically designed for genotyping molecular diagnosis of L1, E1, E2, E6, and E7 genes of the Human Papillomavirus (HPV), this kit facilitates nucleic acid isolation using the DNall VirAll Kit or other approved kits. Verified sample combinations are then added to the master mix primer/probe mix to initiate the reaction. Additionally, the Stark Genotyping HPV Molecular Diagnostic Kit incorporates a second heterologous amplification system to identify potential PCR inhibition. This is detected as an internal control (IC) in the fluorescence channel Cycling Yellow of Real-Time PCR instruments. Through its sampling mechanism, the quality of sample isolation and PCR reaction process can be monitored and controlled to prevent false-negative results. The assay demonstrates a Limit of Detection (LoD) of 5 Copies/5µl for types 16, 18, and 45, 50 Copies/5µl for types 6/11, 51, 56/66, 33/52/58, 35, 59, and 39, and 500 Copies/5µl for types 68 and 31.

Preparation

Cervical screening, Pap smear, Urine, and Paraffin-embedded tissue samples:

Fresh specimens should either be processed immediately according to the sample procedure outlined in the Sample Processing Protocol section or stored frozen at -20°C. Frozen samples must be brought to room temperature before initiating sample processing. Sample Pre-treatment decontaminates the specimen and prepares it for extraction.

DNA Isolation

For nucleic acid isolation, utilize the DNall VirAll Kit or other approved kits.

Before Starting

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

Buffer Preparation

Take out each component from the kit and allow the reagents to equilibrate to room temperature. Before use, briefly vortex the components. The total volume of isolated nucleic acid should be 5µl. Refer to Table 1 for buffer preparation and Table 2 for PCR run instructions.

Table : Preparation of components per single reaction

Pathogenicity

Human papillomaviruses (HPVs) are small double-stranded DNA viruses that infect the cutaneous and mucosal epithelium. Infection by specific HPV types has been associated with the development of cervical carcinoma. HPV targets epithelial cells undergoing terminal differentiation and employs multiple mechanisms to override normal differentiation regulation, producing progeny virions. This leads to morphological changes resulting in noncancerous tumors in the skin, commonly referred to as “Papilloma”.

Human papillomavirus comprises 150 identified strains categorized into various types. Approximately 75% of papilloma types target epithelial cells (cutaneous types), while the remaining 25% (around four types) target mucosal epithelial tissues, identified as mucosal or genital types. Cutaneous types mainly cause noncancerous epithelial warts in different skin areas, particularly in the lips’ epithelial cells, while mucosal types induce genital warts and cancerous tumors, especially cervical cancer.

Low-risk HPV

Low-risk HPV strains include types that cause noncancerous and low-risk warts in genital areas of both women and men. In women, they predominantly manifest in internal genitalia, such as the vagina and cervix. There are 12 types of papillomaviruses with low cancer risk that can induce warts and morphological changes in the genital area: 6, 11, 40, 42, 43, 44, 53, 54, 61, 72, 73, and 81. Types 6 and 11 are responsible for 90% of genital warts.

High-risk HPV

High-risk HPV can lead to cancers of the cervix, vagina, and vulva in women; penis in men; anus; and the back of the throat, including the base of the tongue and tonsils (oropharynx), in both men and women. There are 14 types of high-risk HPV viruses, including: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Approximately 70% of reported cervix cancers are caused by HPV 16 and 18. Additionally, HPV 16 is responsible for 95% of oral, vaginal, and anal cancers. HPV 31 and 45 are considered high-risk cancer strains, contributing to 5%-10% of cervix cancers.

Workstation Preparation

Before usage, all work surfaces, pipettes, and other supplies must be thoroughly cleaned and sanitized. To minimize the risk of nucleic acid contamination, utilize sanitizers such as 70% Ethanol or 10% Sodium Hypochlorite.

Protocol

Figure : preparation of reagents, PCR run, and interpretation of results.

PCR reaction preparation

Table : PCR reaction preparation

Thermal Profile

Table : Thermal profile for Stark Genotyping HPV Molecular Diagnostic Kit

Control Conditions for a Valid PCR Run

A negative control serves as a contamination control. If the magnitude increase of the fluorescence curve in the negative control does not cross the threshold and Ct is less than 35 (Ct<35), it is considered as possible contamination. Strong signals above 35 in the NTC can be PCR artifacts. In such cases, the shape of the curve, typically an S-shaped curve, should be considered for a positive result.

The internal control should yield a positive result for all clinical specimens with Ct ≤ 35, indicating sufficient nucleic acid from the human gene and acceptable sample quality. If the internal control curve has Ct > 37 or lacks Ct, it indicates low sample concentration or inhibitors in the reaction. In such instances, diluting the isolated sample by at least half is recommended. If the test result remains unacceptable upon retesting, obtain a new sample from the patient and repeat the test.

A positive clinical specimen should have Ct ≤ 37 for the gene. If the expected positive reaction, characterized by a typical S-shaped curve, is not achieved, the performed test is deemed unacceptable. Repeat the test following the instructions provided in the kit catalog.

In case of failure of the positive control, determine the reason, take corrective action, and document the results of the corrective action.

Table : Control conditions for a valid PCR Run

The Stark Genotyping HPV Molecular Diagnostic Kit undergoes rigorous testing against predetermined experiments on a lot-to-lot basis to ensure consistent product quality. Accessible results of these experiments are obtainable online by referencing the REF and Lot numbers at http://www.amorph.tech.

Data analysis for each type should be performed separately using a manual threshold. For the detection of L1, E1, E2, E6, and E7 genes, fluorophores FAM (green), Yakima Yellow (Yellow), and ROX/Texas Red (orange) are utilized for Pro1 HPV Mix to Pro4 HPV Mix. The IC gene in Pro2 HPV Mix is detected using the Yakima Yellow (Yellow) fluorophore.

Table : Specific fluorescent channels identified for HPV types.

Table : Stark Genotyping HPV Molecular Diagnostic Kit features and specifications

For bellow information refer to the Product IFU:

• Limit of Detection (LoD) – Analytical Sensitivity
• Cross-reactivity (Analytical Specificity)
• Cross-Reactivity (Clinical Specificity)
• Clinical Evaluation

• Low virus titers in patients’ specimens, improper transportation, and low-quality DNA isolation can lead to false-negative results.
• All related controls should be checked before result interpretation to ensure reliable results. Failure to do so may compromise the accuracy of the results.
• The limit of detection (LoD) of the present kit is demonstrated by Ct ≤ 37, and a typical S-shaped curve must appear for all positive specimens.
• Improper storage conditions can result in false-negative results.
• The product should only be used by personnel specially instructed and trained in in-vitro diagnostics, as individual errors can compromise the accuracy of the results.
• Diagnosis of Human papillomavirus infection is made when test results are positive and accompanied by clinical symptoms. Treatment should be conducted based on diagnostic kit results, medical background, and response to treatment.

Storage

Ensure all reagents are stored away from direct light in darkness at temperatures ranging between -20°C to -25°C. Avoid frequent freeze-thaw cycles. When stored as directed, all reagents maintain stability until the expiration date indicated on the kit box.

Warning and Precautions

-This kit is for in vitro diagnostic use only.

-Material Safety Data Sheets (MSDS) for all products and reagents are available online at http://www.amorph.tech.

-Adhere to laboratory safety protocols diligently.

-Familiarize yourself thoroughly with the guidelines before usage.

-Prohibit 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 steps of procedures, 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 in line with biosafety guidelines. Regularly sanitize desks and laboratory instruments with 70% Ethanol or 10% Sodium Hypochlorite.

-Shield Pro Mixes from sunlight exposure.

-Clean and disinfect specimen spills promptly 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 pertain to the components of the Stark Genotyping HPV Molecular Diagnostic Kit.