Viga Genotyping HPV Molecular Diagnostic Kit

Description

Kit content

Kit Content	25 Preps	100 Preps
Q-ROMAX, 4X	600µl	2400µl
Pro 1 HPV Mix	350µl	1400µl
Pro 2 HPV Mix	350µl	1400µl
Pro 3 HPV Mix	350µl	1400µl
Pro 4 HPV Mix	350µl	1400µl
Positive Control	100µl	200µl
Water (PCR Grade)	150µl	600µl

Description

The Viga Genotyping HPV Molecular Diagnostic Kit technology is an in vitro nucleic acid TaqMan assay with signal amplification using polymerase chain reaction and fluorescent probs (ROX/Texas Red, Yakima Yellow, and FAM) for the genotyping detection of 14 high-risk and two low-risk types of HPV DNA in cervical or vaginal specimens. The HPV types detected by the test are the high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 and 2 low-risk types 6, 11 in human cervical screening, cytology sample, urine, and paraffin-embedded tissue. This diagnostic test kit utilizes the polymerase chain reaction (PCR) and is configured with three-Channels Multiplex Real-Time PCR instruments.

• Detection and simultaneously genotyping of 14 High
• Risk HPV Genotypes and 2 Low Risk HPV Genotypes.
• Endogenous internal control DNA
• High Specificity
• High Sensitivity
• Extensively validated on clinical samples
• Rapid, reliable, comprehensive and cost-effective test.
• Easy work flow
• Compatibility with the most common Real Time-PCR equipment

Specimen collection

Storage and Sample Preparation

Cervical screening, Pap smear, Urine, and Paraffin-embedded tissue sample

The fresh specimen must either be processed immediately as per sample procedure outlined in the section on Sample Processing Protocol or stored frozen at -20°C. Frozen samples must be brought to room temperature before starting sample processing. Sample Pre-treatment decontaminates the specimen and makes it ready for extraction.

Before Start

Take out 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.

Specimen isolation

For viral nucleic acid isolation use, DNall Plus kit (DN983049 or DN983048), DNall Pro (DN983064 or DN983063) or other kits with confirmation of ministry health.

Process

Take out each component from the kit and allow the reagents to equilibrate to room temperature. Before use, vortex components briefly. The whole volume of isolated nucleic acid should be 5µl. Follow table 1 to prepare buffers and table 2 for PCR run.

Table1: preparation of components per single reaction

components	Volume
Q-ROMAX, 4X	6µl
Pro 1 HPV or Pro 2 HPV or Pro 3 HPV or Pro 4 HPV Mix	14µl
Isolated DNA	5µl

 

Table2: Thermal profile for Viga Genotyping HPV Molecular Diagnostic Kit

	Temperature	Incubation Time	Cycle Numbers
Pre-Denaturation	95 °C	1 min	1
Denaturation	95 °C	10 sec	45
Annealing and acquisition on channel Green, Yellow and Orange	57°C	30 sec

 

Interpretation of results

Data analysis for each type should be performed separately using a manual threshold. Use the following table for results interpretation, showing that Pro1 HPV to Pro4 HPV mixes are detectable in identified channels.

For detection of L1, E1, E2, E6 و E7 genes fluorophore FAM (green), Yakima Yellow (Yellow), and ROX/Texas Red (orange) are identified for Pro1 HPV Mix to Pro4 HPV Mix and Yakima Yellow (Yellow) for IC gene in Pro2 HPV Mix.

Table 3: Specific fluorescent channels identified for HPV types.

ProMIX	Green	Yellow	Orange
Pro1 HPV	45	16	18
Pro2 HPV	51	Internal Control	56/66
Pro3 HPV	35/39	33/52/58	6/11
Pro4 HPV	31	68	59

 

A negative control is used as contamination control. The magnitude increase of the Fluorescence curve in the negative control does not cross the threshold. If Ct is less than 35 (Ct<35), it is considered as possible contamination. Strong signals above 35 in the NTC can be PCR artifacts, which in these cases, the shape of the curve can be considered (the S-shaped curve is typical for a positive result).

Internal control should be positive for all clinical specimens at Ct 35 or less than 35, indicating sufficient nucleic acid from the human gene and the sample has acceptable quality.

Internal control curve with Ct>37 or without Ct indicates low sample concentration or inhibitors in the reaction (the isolated sample is recommended to dilute at least ½). If the test result is not acceptable again during the retest, another new sample should be taken from the patient, and the test must be repeated.

A positive clinical specimen should have Ct≤40 for gene.

If the expected positive reaction is not achieved (typical S-shaped curve), the performed test is not acceptable. The test must be repeated based on kit instructions accessible in the kit catalog.

Determine the reason for the failure of positive control, take corrective action, and document correctional action results.

For more information about positive and negative specimens, refer to table 3.

Table 4:  Control conditions for a valid PCR Run

ProMIX	ROX/Texas Red	Yakima Yellow	FAM	results
Pro 1 HPV	+	+	+	Positive:45 Positive: 16 Positive:18
Pro 2 HPV	+	It is not considered	+	Positive:51 Positive:56/65
Pro 3 HPV	+	+	+	Positive:35/39 Positive:33/52/58 Positive:6/11
Pro 4 HPV	+	+	+	Positive:31 Positive:68 Positive:59
Pro 2 HPV	–	+	–	Negative Result
Pro 1 HPV  or Pro 2 HPV  or Pro 3 HPV  or Pro 4 HPV	–	–	–	Invalid results

 

Limitations

• Low virus titers in patients’ specimens, improper transportation, and low-quality DNA isolation can cause false-negative results.
• All related controls should be checked before result interpretation. Otherwise, results are comprisable.
• The limit of detection of the present kit demonstrated Ct≤40, and the typical S shape curve must appear for all positive specimens.
• Improper storage can lead to false-negative results.
• The product is to be used by personnel specially instructed and trained in the in-vitro diagnostics only as individual errors can compromise the results.
• The patient is diagnosed as infected with Human papillomavirus in cases test positive results accompanied with clinical symptoms, so the appropriate treatment is conducted based on diagnostic kits result, medical condition backgrounds, and response to remedy.
• Limit of detection (LoD) determined for types 16, 18/45 is 5 Copies/5µl, for types 6/11, 51, 56/66, 33/52/58, 35, 59, 39 is 50 Copies/5µl and for types 68, 31 is 500 Copies/5µl.

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