Rapid detection of HPV high-risk types 16 and 18 in cervical cancer screening

  • Rapid detection of HPV high-risk types 16 and 18 in cervical cancer screening

    Automated PCR sample preparation using the Sansure diagnostic kit with the ASSIST PLUS pipetting robot

    Human papillomavirus (HPV) infections are known to be a high-risk factor in developing various types of diseases. Crucially, almost all cervical cancer cases are related to oncogenic HPV infections. The disease remains the fourth most common cancer in women, with an estimated 530,000 new cases per year, and approximately 275,000 deaths worldwide. There are several types of high-risk oncogenic human papillomaviruses, including HPV types 16 and 18 that are responsible for around 70 % of cervical cancers worldwide. However, HPV-related cervical cancer could be prevented with regular screening. HPV testing is more sensitive than the Pap smear. As a result, many countries are now considering the use of HPV testing either to complement smear testing or as a primary test. Consequently, the demand for HPV testing is increasing, driving a need for automation while still keeping the process simple. The High-risk Human Papillomavirus DNA Fluorescence Diagnostic Kit (Sansure Biotech Inc.) is a nucleic acid extraction and amplification test for the detection of the high-risk oncogenic HPV types 16 and 18. It does not require time-consuming DNA purification steps prior to the PCR set-up, allowing for faster results compared to most PCR tests as well as increased sample processing capacity.

  • Table of contents

    Human papillomavirus (HPV) infections are known to be a high-risk factor in developing various types of diseases. Crucially, almost all cervical cancer cases are related to oncogenic HPV infections. The disease remains the fourth most common cancer in women, with an estimated 530,000 new cases per year, and approximately 275,000 deaths worldwide. There are several types of high-risk oncogenic human papillomaviruses, including HPV types 16 and 18 that are responsible for around 70 % of cervical cancers worldwide. However, HPV-related cervical cancer could be prevented with regular screening. HPV testing is more sensitive than the Pap smear. As a result, many countries are now considering the use of HPV testing either to complement smear testing or as a primary test. Consequently, the demand for HPV testing is increasing, driving a need for automation while still keeping the process simple. The High-risk Human Papillomavirus DNA Fluorescence Diagnostic Kit (Sansure Biotech Inc.) is a nucleic acid extraction and amplification test for the detection of the high-risk oncogenic HPV types 16 and 18. It does not require time-consuming DNA purification steps prior to the PCR set-up, allowing for faster results compared to most PCR tests as well as increased sample processing capacity.

Key benefits

  • Automating VOYAGER adjustable tip spacing pipettes on the ASSIST PLUS pipetting robot provides a fast, efficient and error-free, sample preparation method for 96 samples.
  • The system increases sample throughput, eliminates the risk of reformatting errors and minimizes manual intervention.
  • The method described in this application note is ready for molecular diagnostic labs to use, making the implementation of high-risk oncogenic HPV testing fast and straightforward, with no additional set-up steps or programming required by the user.
  • The Sansure High-risk Human Papillomavirus DNA Fluorescence Diagnostic Kit is an established product that enables DNA testing with a 15-minute lysis, minimizing the time until results are provided to patients.

Overview: How to automate the pipetting steps of the Sansure High-risk Human Papillomavirus DNA Fluorescence Diagnostic Kit

The ASSIST PLUS pipetting robot is used to automate the pipetting steps of the Sansure High-risk Human Papillomavirus DNA Fluorescence Diagnostic Kit. 1 ml of each sample is collected and placed in 1.5 ml microcentrifuge tubes before centrifugation for 5 minutes at 12,000 x g. The ASSIST PLUS pipetting robot operates a VOYAGER 8 channel 50 μl adjustable tip spacing pipette with 125 μl Sterile, Filter GripTips.

Overview of the steps:

1. Lysis of the first sample series (1-48)
2. Lysis of the second sample series (49-96)
3. Addition of the master mix

Downloads: Protocols and reports for automated sample preparation for PCR

The ASSIST PLUS pipetting robot reformats and prepares the samples for PCR using a pipetting program generated with the VIALAB software.

Experimental set-up

Prepare the deck of the pipetting robot as follows (Figure 1):

Deck position A:

Dual reservoir adapter containing a 25 ml divided reservoir with SureFlo™ anti-sealing array.

Deck position B:

96 well PCR plate or tubes placed on an INTEGRA PCR cooling block in landscape orientation.

Deck position C:

INTEGRA rack for 1.5 ml microcentrifuge tubes containing the patient samples.

General set-up for the sample preparation protocol.
Figure 1: General set-up for the sample preparation protocol.

Step-by-step procedure

96 samples can be prepared in less than 30 minutes using the configuration described in this application note.

1. Lysis of the first sample series (1-48)

The lysis buffer is added to the first series of samples (1-48) and mixed. After a delay, the samples are transferred to the 96 well PCR plate.

Fill the 10 ml compartment of the divided reservoir with 8 ml of the lysis buffer as shown in Figure 1. Select and run the VIALAB program ‘HPV 96 well’. The ASSIST PLUS pipetting robot transfers 50 μl of the lysis buffer from the reservoir into the 1.5 ml microcentrifuge tubes and mixes the samples by aspirating and dispensing 4 times. The GripTip pipette tips are replaced at the end of each mix cycle to avoid cross-contamination from tube to tube. After completion of this step, a 15-minute delay is required to ensure complete lysis of the cells. The ASSIST PLUS pipetting robot then transfers 10 μl of the first series of lysed samples into the first half of the 96 well PCR plate on the INTEGRA cooling block (Figure 2).

Tips:

  • Sterile, Filter GripTips are used to prevent aerosols from contaminating the pipette and samples.
  • The PCR cooling block is used as a support for a 96 well PCR plate. It provides passive cooling of the samples when required by the protocol.
Sample distribution on the 96 well PCR plate.
Figure 2: Sample distribution on the 96 well PCR plate.

2. Lysis of the second sample series (49-96)

The lysis buffer is mixed with the second series of samples (49-96). After lysis is complete, the samples are transferred into the 96 well PCR plate.

The pipette prompts the user to change the INTEGRA microcentrifuge tube rack to the second series of samples (49-96). The ASSIST PLUS then transfers and mixes the lysis buffer as described in step 1. Afterwards, the plate is incubated for 15 minutes. The lysed solution is then transferred into the second half of the 96 well PCR plate on the INTEGRA cooling block (Figure 2).

3. Master mix addition

The master mix is added into the 96 well PCR plate.

The ASSIST PLUS pipetting robot prompts the user to fill the 5 ml compartment of the 25 ml divided reservoir with 5 ml of the master mix (Figure 3). The ASSIST PLUS pipetting robot transfers 39 μl of the master mix from the reservoir into the 96 well PCR plate. The solution is mixed thoroughly by aspirating and dispensing 3 times.

Tip:

  • Using the 5 ml compartment of a 25 ml divided reagent reservoir with SureFlo™ anti-sealing array allows a very low dead volume and minimizes the loss of expensive master mix.
Deck set-up for the master mix transfer step.
Figure 3: Deck set-up for the master mix transfer step.

Remark

Partial plates:

A separate VIALAB program for processing of 48 samples (one 48-test High-risk Human Papillomavirus DNA Fluorescence Diagnostic Kit) is available for download. The program can also be adapted to a different number of samples, giving laboratories total flexibility to meet current and future demands.

Conclusion

  • The Sansure High-risk Human Papillomavirus DNA Fluorescence Diagnostic Kit, in combination with the ASSIST PLUS pipetting robot, supports early cervical cancer prevention by offering a simple, fast and automated solution for the detection of HPV DNA.
  • 96 samples can be prepared in less than 30 minutes using the configuration described in this application note.
  • The small footprint of the affordable ASSIST PLUS pipetting robot means that it can easily be placed under a laminar flow cabinet, protecting lab personnel from contamination risks.
  • The pipetting steps of the VIALAB program have been optimized to ensure the simplest use of the High-risk Human Papillomavirus DNA Fluorescence Diagnostic Kit and the best diagnostic results.
  • The VOYAGER adjustable tip spacing pipette eliminates the risk of errors when reformatting patient samples from tubes to plates.
  • The ASSIST PLUS pipetting robot, together with the Sansure High-risk Human Papillomavirus DNA Fluorescence Diagnostic Kit, offers a ready-to-use solution that is easy to implement, with no additional setup required or programming skills necessary.

September 2020, by

  • Jan-Peter Baldin, PhD

    Product Manager

    Email

Any questions? I'm happy to help!

  • Stephanie Stursberg

    Product Manager

    Email

Instruments and accessories

ASSIST PLUS, Pipetting Robot VOYAGER, 8 Channel, 50 µl GripTip, 125 µl, Sterile, Filter Rack for 1.5/2 ml microcentrifuge tubes PCR 96 well cooling block Dual Reservoir Adapter Divided Reagent Reservoir, Sterile, SureFlo anti-sealing array, 25 ml, PP Greiner Bio-One: 96 Well Polypropylene Microplates SANSURE Biotech: High-risk Human Papillomavirus DNA Fluorescence Diagnostic Kit

ASSIST PLUS, Pipetting Robot

INTEGRA has developed the ASSIST PLUS pipetting robot to streamline routine pipetting tasks at an affordable price. Using INTEGRA electronic multichannel pipettes, the system:

  • automates pipetting tasks,
  • eliminates physical strain and
  • ensures superior reproducibility and
  • error free pipetting.

Part No. 4505

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ASSIST PLUS Pipetting Robot

VOYAGER, 8 Channel, 50 µl

VOYAGER pipettes allow the tip spacing to expand anywhere between 4.5 mm and 33 mm at the push of a button.

  • Single handed operation leaves the other hand free to handle labware.
  • On the fly access to up to 3 user-defined tip spacings.
  • These user defined tip spacings are saved. No need to memorize spacings of different labware formats.

Part No. 4726

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VOYAGER, 8 channels, 50μL

GripTip, 125 µl, Sterile, Filter

GripTip pipette tips perfectly match the multi-lobe tip fitting, snapping firmly on during loading. This guarantees a perfect seal on every tip, preventing them from loosening, leaking or completely falling off. All tips are precisely aligned horizontally, enabling accurate touch-offs, even when pipetting with 384 tips.

Part No. 4425

Learn more about GripTips pipette tips for Benchtop Pipetting Systems
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Rack for 1.5/2 ml microcentrifuge tubes

Hold up to 48 tubes on removable carriers for easy loading and storage.

Part No. 4540

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Rack for 1.5/2 ml microcentrifuge tubes

PCR 96 well cooling block

Aluminium 96 and 384 well PCR cooling blocks that keep reagents chilled during sample preparation in a PCR plate. The cooling blocks feature a SBS footprint.

Part No. 6250

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PCR cooling block

Dual Reservoir Adapter

The dual reservoir adapter makes this system ideal for assays that required sequential addition of several low volume reagent mixes. This adapter holds two 10 ml, 25 ml or divided reagent reservoirs side by side in an aluminum frame, and can be pre-chilled to keep reagents cool during processing.

Part No. 4547

Find out more about ASSIST PLUS accessories
Dual reservoir adapter with 2 didived reservoirs

Divided Reagent Reservoir, Sterile, SureFlo anti-sealing array, 25 ml, PP

Lowest Dead Volume On The Market!

25 ml divided reservoirs are comprised of 5 and 10 ml compartments, while still nesting comfortably in the standard 25 ml reusable, sturdy base. 
Each reservoir features the SureFlo™ anti-sealing array and hydrophilic surface treatment providing especially low dead volumes.

Part No. 4356 / 4357

Find out more about Divided Reagent Reservoirs
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Greiner Bio-One: 96 Well Polypropylene Microplates

SAPPHIRE MICROPLATE, 96 WELL, PP, FOR RT PCR, WHITE, HALF-SKIRT, FOR LIGHT CYCLER, 10 PCS./BOX

Source: Website Greiner Bio One

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SANSURE Biotech: High-risk Human Papillomavirus DNA Fluorescence Diagnostic Kit

This diagnostic kit is an in vitro nucleic acid amplification test for the detection of high-risk papillomavirus (HPV)(Type 16 and18) DNA in reproductive tract secretions. It is intended for use as an aid in diagnosing an HPV (Type 16 and18) infection, and the early screening of cervical cancer

Source: Website SANSURE BIOTECH INC.

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High-risk Human Papilomavirus DNA Fluorescence Diagnostic Kit (PCR-Fluorescence Probing)

Customer’s voice

The Assist Plus has freed up staff time to do other jobs, while giving consistent reliable and precise results. Add to that the incredible ease of writing protocols, even if you have very little experience with robots. It's quiet, and just does what you expect it to do.

Tracey Van Stijn, AgResearch