Quantifiler® HP and Trio Kits

Both kits are available in a 400-reaction size, similar to Quantifiler® Human, Male Y, and Duo kits. We have enhanced our kit packaging to help laboratories minimize the potential for contamination and facilitate automation capabilities. Instead of one bottle containing the reaction mix, we now have four 100-reaction mix tubes with sufficient overfill, which makes it easier to use one tube per 96-well quantification plate.

We have also packaged the DNA standard in a separate box. The boxes for both the reaction mix and the DNA standard are housed together with an outer sleeve that has peel-and-stick barcode labels similar to those on GlobalFiler™ kits. This makes it easier to track reagent lot numbers and to separate reagents for storage upon arrival at the lab.

There is an additional large autosomal (LA) target that has been added as part the degradation index feature in the Quantifiler® Trio kit. This marker plus the improved small autosomal (SA) and male targets make three different quantification targets in the Trio kits, whereas the Quantifiler® Duo kit has only two targets: one autosomal and one male.

The new degradation index functionality in the Quantifiler® Trio kit enables a more comprehensive view of sample quality (both degradation and inhibition), and the kit also has increased sensitivity and robustness to better correlate with more recently released Applied Biosystems® STR chemistries, such as those in the Identifiler® Plus, NGM SElect™, and GlobalFiler™ PCR Amplification Kits.

The “HP” stands for “Human Plus” because this kit is intended as an upgrade to the Quantifiler® Human kit. Like the Quantifiler® Human kit, the new Quantifiler® HP kit detects and quantifies human DNA (without a male target), but the new kit also includes a large autosomal (LA) target to enable degradation index functionality and more comprehensive sample quality assessment. It also features increased sensitivity and robustness to better correlate with more recently released Applied Biosystems® STR chemistries such as those in the Identifiler® Plus, NGM SElect™, and GlobalFiler™ PCR Amplification Kits.

The quality index is a measurement of both sample degradation (degradation index) and inhibition (IPC). A degradation index refers to the data observed when a sample displays a decrease in the measured amount of large DNA fragments compared to small DNA fragments. In the Quantifiler® HP and Trio kits, the degradation index is measured by comparing the quantification results for the small autosomal (SA) and large autosomal (LA) targets, and is used as a general indicator of whether large DNA fragments may perform poorly relative to small DNA fragments in STR reactions.

Forensic casework samples commonly contain inhibitors that can interfere with PCR amplification. The Quantifiler® HP and Trio kits have been designed with an inhibitor tolerance that is ~5x higher than that of the Quantifiler® Duo kit, which more closely correlates with more recently released, highly robust Applied Biosystems® STR chemistries such as those in the Identifiler® Plus, NGM SElect™, and GlobalFiler™ PCR Amplification Kits.* The degradation index can be affected by both the degree of degradation of the LA target and the presence of PCR inhibitors, so it’s important to evaluate the degradation index in conjunction with the Internal PCR Control (IPC) results in the HID Real-Time PCR Software, to determine the overall sample quality and the optimal approach to maximize data recovery during STR analysis.*

With an approximate cycling time of only 60 minutes, these new kits are ~33% faster than previous-generation Quantifiler® chemistry—and they can provide more information regarding sample quantity and quality in a single reaction. The new quality index provides a more comprehensive view of sample quality (both degradation and inhibition) to facilitate up-front decisions on how to proceed to the STR reaction for optimal data recovery.

Also, improved sensitivity at sub-picogram levels of DNA enables more accurate identification of samples that contain insufficient DNA to proceed with STR and/or Y-STR analysis, saving valuable time and resources in the lab.

Each new kit includes updated protocols for flexible sample processing; a reduction in standard curve points, enabling more samples to be processed per run; and faster time-to-results with a 33% reduction in cycling times. Key features of the new HID Real-Time PCR Software v1.2** include configurable Quantifiler® templates, data quality assessment, and integrated sample normalization/STR reaction setup tools for simplified, faster sample processing from quantification to STR analysis.

Internal validation data have shown that the Quantifiler® HP and Trio kits can detect at the limit of ~1/10 of a cell equivalent (<1 pg/µL), which is a significant (>6x) increase in sensitivity compared to previous-generation Quantifiler® kits. This is achieved through the use of multicopy target loci that provide much greater detection sensitivity than single-copy assay targets. The targets in the Quantifiler® HP and Trio kits have been selected to maximize quantification sensitivity for challenging, low-concentration samples, including an improved male target (in the Trio kit) to deliver more accurate results for samples containing low concentrations of male DNA in the presence of high amounts of female DNA.

In addition, these multicopy target loci were carefully selected to minimize copy number variation between individuals and population groups, through the use of robust bioinformatics tools and extensive R&D testing. The improved sensitivity at sub-picogram levels of DNA, combined with high reproducibility, enables laboratories to more accurately and confidently identify and screen out samples that may not contain DNA sufficient for STR and/or Y-STR analysis—saving valuable time and resources in the lab.

The increased robustness (sensitivity, inhibition detection, and M:F ratio) of the Quantifiler® HP and Trio chemistries was primarily designed to better correlate with downstream results when used in conjunction with the more recently released, highly robust Applied Biosystems® STR kits. These include Identifiler® Plus, NGM SElect™, Yfiler® Plus, and GlobalFiler™ kits.

The Quantifiler® Human, Male, and Duo DNA Quantification Kits correlate more closely with the legacy Identifiler®, Profiler®, COfiler®, SGMPlus®, and Yfiler® kits in terms of inhibition detection but do not offer any information on DNA degradation. The HID Real-Time PCR Software v 1.2 supports the setup and analysis of all currently available Applied Biosystems® STR kits and Quantifiler® chemistries.

Both the Quantifiler® HP and Trio kits have been designed with two human autosomal targets: a small autosomal (SA) target and large autosomal (LA) target. Both targets provide quantitative results for a sample; however, with increasing degradation, longer-target amplicons tend to decrease disproportionately relative to shorter-target amplicons. The relative amounts of LA amplicons and SA amplicons assist users in identifying possible sample degradation. This result is referred to as the degradation index.

The small autosomal (SA) target is used as the primary target marker for quantification, as DNA degradation and inhibition may impact the quantification of the large autosomal (LA) target.

Yes, we have improved the homogeneity and stability of the DNA standard by using lower-molecular-weight pooled genomic male DNA at a lower concentration (100 ng/μL vs. 150 ng/µL for the Duo kit standard) as well as improved manufacturing QC processes. To increase stability of the prepared standard dilution series, we recommend storing the prepared standards in low-bind tubes—for example, Applied Biosystems® Nonstick RNase-free Microfuge Tubes, 1.5 mL, Catalog # AM12450. Diluted DNA quantification standards can be stored up to 2 weeks at 2–8°C.

Yes, the recommended dilution series for both the Quantifiler® HP and Trio Kits has been reduced from 8 concentration points to 5 points. This improvement over the previous kits was made in response to requests for more informative points on the standard curve combined with higher sample throughput. This change allows for approximately 6% more sample processing per 96-well plate. (Note: an optional 100 ng/μL standard point can be added for processing higher-concentration samples. Consult the user guide for more information.)

By subtracting the male DNA concentration from the human DNA concentration, the concentration of female DNA can be approximated.

We evaluated male:female DNA ratios up to 1:4,000 with 20 pg/µL male DNA and were able to accurately quantify the male and female DNA fractions.

HID Real-Time PCR Analysis Software v1.2

Yes, the new software runs on both Windows® XP SP3 and Windows® 7 SP1 (64-bit and 32-bit).

The new Quantifiler® Trio and HP kits and HID Real-Time PCR Analysis Software v1.2 have been validated to perform on the Applied Biosystems® 7500 Real-Time PCR System.***

The software is designed to run on both the instrument computer and stand-alone desktop computers, and they must meet the following system requirements:

Recommended requirements

Pentium® 4 or compatible processor, 2.0 GHz (minimum 1.2 GHz)

  • 1 GB RAM
  • One hard drive with 10 GB available
  • 20/48X IDE CD-ROM drive
  • USB v2.0 (minimum v1.1)
  • Ethernet network interface adapter (10BASE-T)

Compatible operating systems

  • Microsoft Windows® 7
  • Microsoft Windows® XP, Service Pack 3

Firmware

  • Gv2.10 for 7500

The HID Real-Time PCR Software v1.2 has been fully validated for human identification and is Microsoft Windows® 7–ready. The key workflow features include configurable Quantifiler® templates as well as instrument calibration, data quality assessment, and integrated sample normalization/STR reaction setup tools for simplified, faster sample processing from quantification to STR analysis. It can automatically calculate the degradation index, male:female mixture ratio, and dilution values, and includes a number of customizable data quality flags for rapid data review. It also has the ability to link your quantification workflow between automated liquid handlers and the Applied Biosystems® 7500 Real-Time PCR System to enable faster, more streamlined reaction setup. The HID Real-Time PCR Software uses different analysis algorithms than the SDS v1.2.3 software. In our internal comparison testing, the HID Real-Time PCR Software generated CT values with statistically significant differences compared to the SDS v1.2.3 software.

In addition to a new degradation index and Microsoft Windows® 7 support, the new software has been optimized for use with the Quantifiler® Trio and HP kits along with our latest STR kits, such as the GlobalFiler™ PCR Amplification Kit.

Yes, the new HID Real-Time PCR Analysis Software v1.2 can read and reanalyze data files generated by the previous version of HID Real-Time PCR Analysis Software.

The Quantifiler® Trio and HP Kits were developed and validated using the HID Real-Time PCR Software v1.2.  The software ensures the optimal performance of new Quantifiler® Trio and HP kit-specific functionality such as the automatic calculation of M:F ratios, Degradation Index and streamlined setup for the latest STR analysis kits including the Identifiler Plus™, NGM Select™ and GlobalFiler™ kits.

The software was designed as an integrated solution with the 7500 Real-Time PCR System for human identification and the latest-generation Applied Biosystems® STR kits and Quantifiler® chemistries. It can also be installed as stand-alone software for analysis.

No, the software is supported by both Microsoft Windows® XP and 7, so you can continue running the instrument on the current XP platform. However, since Microsoft has stopped supporting the XP platform, we will no longer be able to provide you a replacement computer on Windows® XP, since all future purchases of the 7500 Real-Time PCR System for Human Identification will be shipped with Microsoft Windows® 7.

No. If your instrument computer is one of the validated models—Dell XE tower, Dell E6520 or E6530—you do not need to acquire a new computer.

Automation

Robotic integration is enabled by the automation-friendly bundling of the Quantifiler® Trio and HP kits combined with the HID Real-Time PCR Analysis Software v1.2, which has been designed to facilitate automation with commonly used robotic platforms. In addition, our Human Identification Professional Services (HPS) team has extensive expertise in developing customized robotic scripts and conducting on-site verification and validation testing to help you achieve optimal performance with a wide range of platforms. The HPS team has already developed scripts for the Quantifiler® Trio and HP kits on several of the most common robotic platforms, and would be happy to consult with your lab on how to efficiently implement these solutions into your workflow. Please contact your local human identification (HID) account manager to learn more.

* For Forensic or Paternity Use Only.
** For Research, Forensic, Paternity and Cell Line Authentication. Not for use in Diagnostic and Therapeutic applications.
*** When used for purposes other than human identification, the instruments cited are for Research Use Only. Not for use in diagnostic procedures.