Technology

AmpaSand® Bead technology

AmpaSand beads are silica microspheres that have been extensively modified using Genera’s proprietary technology to create identifiable clusters of beads which can be distinguished in a conventional flow cytometer (a standard piece of laboratory equipment). The beads act as a microscale platform upon which a variety of biochemical reactions can take place, such as the binding of markers for disease.

The ability to identify individual clusters of beads within a larger pool facilitates the “multiplexing” of tests. Multiplexing is the term used to describe the conducting of tests for several different markers simultaneously. This can provide more information, and increases the efficiency of testing. For example, several different sexually transmitted diseases (STDs), such as HPV, Chlamydia and Gonorrhoea may be tested for using just a single multiplexed test, rather than three separate tests. More broadly, it opens opportunities for Genera to develop a range of sophisticated diagnostic products.

AmpaSand Bead Technology

AmpaSand bead systems offer advantages over latex bead technologies. Silica beads are stable to 1,000oC, and this heat stability permits certain processes, some of them proprietary to Genera, that would not be possible with latex beads (for example high-stringency DNA Hybridisation and single-tube PCR/Hybridisation processing).

 

Solid Phase PCR

Solid phase PCR amplification of targets utilizes specific primers (‘Probe’ primers) conjugated to AmpaSand Beads and red fluorescently labeled aqueous (Reverse) primers (see figure below). Additional unlabeled aqueous (Forward) primers are included at limiting concentrations to enrich the target and promote the solid phase PCR. As a consequence of the solid phase amplification process, the red reverse primers become incorporated into the growing PCR product and thus increase the red color intensity associated with the AmpaSand Bead. After PCR, beads are analyzed in a flow cytometer, which allows the differentiation by size and green color intensity of all the target-defined bead populations. A significant shift in the red fluorescence of a bead population is indicative of a successful solid phase PCR amplification event. Positive “calls” for target pathogens occur when the median red fluorescence of a bead population exceeds its experimentally determined threshold level. QPlots, Genera Biosystems’ proprietary software, uses these thresholds, as well as data from internal calibration controls on each test plate, to make objective calls for each pathogen for each clinical sample. The QPlots results are stored electronically, as well as printed in easy to read format.

Solid Phase Amplification
The PCR reaction is essentially a nested PCR containing AmpaSand Beads with an immobilized internal forward primer. This primer contains a linker region that allows for covalent attachment to the bead as well as the active amine moiety for attachment of a fluorescent tag. Genera uses a Green fluorescent tag for bead marking for its current diagnostic kits. The liquid phase forward primer and fluorescently labeled reverse primers act as external primers. As the PCR progresses, the forward strand grows from the immobilized primer (shown as black dashed strand) and the fluorescently labeled reverse primer becomes incorporated into the complementary strand (red dashed strand). This strand gives the bead the color for determination of positive or negative: the more complement, the brighter red the bead and a positive call. If no Target is present in the sample, there is no amplification and thus no change of color on the bead.

IVD Products

PapType® CE Certified (CE-IVD)

PapType is a highly competitive molecular diagnostic test for the simultaneous detection and genotyping of the 14 high-risk and 2 low-risk types of human papillomavirus (HPV), the viruses that cause 99.7% of cervical cancer in women and genital warts.

The test begins with the collection of cervical cells from a patient in either liquid cytology media, or swabs. From these cells, DNA is extracted and a small portion of this purified DNA is used in a Polymerase Chain Reaction (PCR) that includes Genera’s beads and buffers, as well as thermostable taq polymerase. Genera’s beads are supplied in a PCR-ready 96-well plate. Each sample reaction well within the plate contains a bead pool in which HPV type-specific oligos are covalently attached to specific bead types. Each of the specific bead types in the pool corresponds to one of 17 targets, including:

  • 14 high risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68),
  • 2 low risk HPV types (6, 11), and;
  • an internal human control.

In addition to the reaction wells in each plate, there are 8 control wells. These controls include a water control, a negative control (Jurkat cell genomic DNA) as well as a positive HPV type 18 control (HeLa cell genomic DNA). Additional controls are used to calibrate the fluorescence levels of each plate.

(PCR reactions are completed by adding sample DNAs to an enzyme + primer mix in the sample reaction wells. The enzyme + primer mix contains taq polymerase as well as a red labeled fluorescent PCR primer. After set-up, the plate containing the samples and controls is then placed in a thermal cycler and the cycling is started. If a patient sample contains HPV, DNA will grow on the appropriate bead type by extension of the type specific primer on each bead. As the immobilized strand grows, the bead becomes colored red due to the concomitant growth of the red-labeled reverse complement.

After cycling, the plate is washed to remove unincorporated fluorescence and each sample is analyzed by flow cytometry. The flow cytometry data is analyzed by Genera’s software, QPlots. This analysis results in the 3 possible calls for each sample, including: POS, the detection and identification of specific HPV type(s); NEG, a negative call for patients with no detectable virus; and IND, indeterminate for those samples for which no positive or negative call is possible, due to assay problems such as insufficient DNA.

 

RTIplex CE Certified (CE-IVD)

RTIplex is an automated, high-throughput assay for the simultaneous detection of 12 viral and 3 bacterial respiratory pathogens. The RTIplex kit is used for the high throughput, qualitative detection and identification of fifteen respiratory analytes from nasopharyngeal swab specimens. The viral and bacterial targets identified by RTIplex are shown in Table below:

 

Pathogen (or Control) Target Gene
InfluenzaA (InfA) Segment 7 Membrane Protein (MP)
InfluenzaB (InfB) Segment 4 Hemagglutinin (HA)
InfluenzaA (InfA/H1N1) Segment 4 Hemagglutinin (HA)
InfluenzaA (InfA/H5N1) Segment 4 Hemagglutinin (HA)
Respiratory Syncytial Virus subtypes A & B (RSV) Polymerase (POL)
Parainfluenza 1 (HPIV1) Hemagglutinin-Neuraminidase (HN)
Parainfluenza 2 (HPIV2) Hemagglutinin-Neuraminidase (HN)
Parainfluenza 3 (HPIV3) Hemagglutinin-Neuraminidase (HN)
Parainfluenza 4 (HPIV4) Phosphoprotein (P)
Human Metapneumovirus (hMPV) Matrix Protein 2
Adenovirus B & E subtypes (Adeno) Hexon Capsid Protein
Rhinovirus (RhV) 5’ Untranslated Region (5’UTR)
Bordetella pertussis (B. pertussis) Insertion Sequence 481 (IS481)
Chlamydophila pneumonia (C. pneumo) Major Outer Membrane Protein (MOMP)
Mycoplasma pneumonia (M. pneumo) P1 Cytadhesin
MS2 RNA Control Matrix Protein
Human Control Myosin Alkali Light Chain, Cardiac Isoform (MYL3)

 

Typically, total nucleic acid is extracted from respiratory specimens (such as throat swabs) and is subjected to a solid phase PCR using an optimised pool of primers specific for target RTIplex analytes. To simplify this process, Genera Biosystems provides a heat-sealed Detection Plate in which each well contains a 5 µL aliquot of the RTIplex; bead-pool. Reactions are rapidly assembled by dispensing 10 µL of a RT-PCR premix followed by 10 µL of Unknown/Control samples into each well. The Detection Plate is then sealed and placed on the thermocycler. After cycling, the plate is washed to remove unincorporated fluorescence and each sample is analyzed by flow cytometry. The flow cytometry data is analyzed by Genera’s software, QPlots.

To allow an assessment of sample extraction efficiency, sample integrity and nucleic acid stability throughout the assay procedure, the RTIplex Kit includes a sample of purified RNA derived from a short segment of the MS2 Phage (MS2 RNA Control). This RNA is designed to be co-extracted with clinical samples and ultimately detected in each sample following amplification and processing on the Detection System.

To further assess sample quantity and quality, the RTIplex assay has been designed to confirm the presence of a human-specific gene that codes for the cardiac isoform of the alkali myosin light chain protein (MYL-3) within extracted clinical specimens. Successful detection of a known amount (approximately 50 cell equivalents (c.e.) of chromosomal DNA) of this Human marker within each sample serves to confirm that sufficient nucleic acid has been recovered from the clinical specimen to allow execution of the RTIplex test. A Negative result for the internal human control indicates either insufficient or impure nucleic acid has been recovered from the clinical specimen and a retest is required.

Development Pipeline

Genera’s currently has 3 additional AmpaSand MDx assays in its development pipeline of which an overview of 2 of these are provided below. Our 3rd assay in our development pipeline remains confidential.

Genera aims to continue to collaborate and partner with existing pathology customers as well as well credentialled global IVD companies to accelerate the expansion of our development pipeline to 10 or more high value AmpaSand MDx assays over the next few years.

 

STIplex

STIplex is a 5 plex multiplex assay for common sexualy transmitted infections that enables the detection and discrimination of five pathogens (Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV) and Herpes Simplex Viruses 1 & 2 (HSV-1 & HSV-2) underlying a variety of common sexually transmitted diseases (STDs). A majority of STDs are asymptomatic and some have high prevalence rates. If left untreated, they can become a major cause of diseases such as PID (Pelvic inflammatory disease), extra uterine pregnancy, infertility, chronic pelvic pain, cervicitis and urethritis.

Recently the US Centers for Disease Control and Prevention updated (June 05, 2015) its sexually transmitted diseases treatment guidelines to help healthcare professionals better manage the more than 20 million cases of STDs in the US annually.

The Sexually Transmitted Diseases and Treatment Guidelines, 2015 provides the first update since 2010. It notably includes recommendations for use of nucleic acid amplification tests for Trichomonas vaginalis (which is a STIplex target) and routine trichomonas screening for high-risk populations.

 

STIplex Bead Map

STIplex Bead Map

Viral Targets

  • Trichomonas vaginalis
  • Chlamydia trochomatis
  • Neisseria gonorrhea
  • Herpes Simplex Virus-1
  • Herpes Simplex Virus-2

Enzyme

  • AmpliTaq Gold

Controls

  • Internal Human Control (single copy MYL3 target)

Instrumentation

  • Leading IVD Cytometer
  • Blue and Red lasers

 

BBVplex

BBVplex is a 5 plex multiplex assay for common bloodborne sexualy transmitted infections that enables the detection and discrimination of five pathogens HIV-1 Group M, HIV-1 Group O, HIV-2, Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV).

HCV infection is the most common chronic bloodborne infection in the United States, with an estimated 2.7 million persons living with chronic infection.

Studies of HCV transmission between heterosexual or homosexual couples have yielded mixed results, but generally have found either no or very minimally increased rates of HCV infection in partners of persons with HCV infection compared with those whose partners are not HCV-infected. However, data indicate that sexual transmission of HCV can occur, especially among persons with HIV infection.

All persons with HCV for whom HIV and HBV infection status is unknown should be tested for these infections. [1] As with our approach with our PapType simultaneous HPV genotyping assay Genera’s approach with the design of our BBVplex assay provides a unique opportunity to improve economics for all stakeholders whilst also delivering improved patient outcomes.

 

BBVplex Bead Map

BBVplex Bead Map

Viral Targets

  • HIV-1 Group M (Major): >90% of HIV-1 cases
  • HIV-1 Group O (Outlier): predominantly West Africa
  • HIV-2
  • Hepatitis B Virus (HBV)
  • Hepatitis C Virus (HCV)

Enzyme

  • TaqMan Fast 1-Step

Controls

  • Internal Human Control (single copy MYL3 target)
  • Internal RNA recovery/stability Control (MS2)

Instrumentation

  • Leading IVD Cytometer
  • Blue and Red lasers

 


[1] Source : Centre for Disease Control and Prevention MMWR / June 5, 2015 / Vol. 64 / No. 3

 

Trials and Studies

Journal of Clinical Virology

Individual detection of 14 high risk human papilloma virus genotypes by the PapType test for the prediction of high grade cervical lesions. View article here

Papillomavirus Research, May 2016 – Need for expanded HPV genotyping for cervical screening

Prevalence of high-risk human papilloma virus genotypes and associated risk of cervical precancerous lesions in a large U.S. screening population: data from the ATHENA trial, April 2015.

Comparison of seven tests for high-grade cervical intraepithelial neoplasia in women with abnormal smears: the Predictors 2 study

Royal Women’s Hospital Clinical Trial

The Royal Women’s Hospital Clinical Trial – Comparison of PapType to Digene Hybrid Capture 2, Roche Linear Array and Amplicor for Detection of High-Risk Human Papillomavirus Genotypes in Women with Previous Abnormal Pap Smears (Journal of Clinical Microbiology) paper. View article here

Comparison of Manual and Automated PapType in Clinical Samples – Wolfson Institute

London Clinical Trial – Detection of HR HPV genotypes in abnormal cervical smears. View article here

Enhanced Solid Phase PCR

Enhanced Solid Phase PCR: Mechanisms to increase priming by solid support primers paper. View article here

Wolfson Institute of Preventive Medicine – Cancer screening

 

RTIplex Clinical Validation Data

1,983 patients performed at Sonic Healthcare in Brisbane and Sydney

Targets Target Abbreviations Genera RTIplex Sonic
SNP
In-House RT/PCR using Roche LC480 and *Qiagen Virus mastermix or †Roche “Probe Master” master mix
DHM
(† = Seegene OneStep Flu A/B Type; * = Seegene Anyplex II RV16)
InfluenzaA InfA ✔ * ✔ † *
InfluenzaA (H1N1) H1N1 NO ✔ †
InfluenzaA (H5N1) H5N1 NO NO
InfluenzaB InfB ✔ * ✔ † *
Respiratory Syncytial Virus (A & B) RSV ✔ * ✔ *
Human Parainfluenza1 HPIV1 ✔ * ✔ *
Human Parainfluenza2 HPIV2 ✔ * ✔ *
Human Parainfluenza3 HPIV3 ✔ * ✔ *
Human Parainfluenza4 HPIV4 ✔ * ✔ *
Human Metapneumovirus hMPV ✔ * ✔ *
Adenovirus (B,C, & E) Adeno ✔ † ✔ *
Rhinovirus RhV NO ✔ *
Bordetella pertussis B.pertussis ✔ † ✔ Simplexa BP kit
Chlamydophila pneumoniae C.pneumo NO NO
Mycoplasma pneumoniae M.pneumo ✔ † NO

 

Target x Target Concordance Summary

Target Sonic + Discordant Genera + Discordant Concordant Total Number of Targets % Concordance % Relative Sensitivity (RTIplex / Sonic)
M.pneumo 0 0 928 9288 100.00 100.00
Adeno 6 0 423 429 98.60 83.33
RSV 1 10 114 125 91.20 107.83
hMPV 4 3 53 60 88.33 98.25
HPIV1 0 0 10 10 100.00 100.00
HPIV2 0 2 7 9 77.78 128.57
HPIV3 0 1 19 20 95.00 105.26
HPIV4 0 2 25 27 92.59 108.00
InfA 0 3 35 38 92.11 108.57
InfA/H1N1 0 2 22 24 91.67 109.09
InfB 1 1 25 27 92.59 100.00
B.pertussis 1 8 38 47 80.85 117.95
RhV 5 5 5 15 33.33 100.00
NEG Samples 58 9 1917 1983 96.67 96.96
POS Calls 18 40 391 420 97.23 104.65

 

Total POS Concordance = 97.23%

Total NEG Concordance = 96.67%

% Relative Sensitivity = # Genera Calls#Sonic Calls = 104.65%

 

Target x Target Concordance Summary

Total Sample Concordance = # Concordant SamplesTotal Samples = 97.03%
Sample x Sample Concordance
# of concordant samples are shown in yellow in diagonal. Discordant classes are shown in red in margins. Column and Row (and Overall) Totals are shown in green.

Collaborations

An Active Partnering Program

Our goal is to increase the pace of commercialization for our existing AmpaSand molecular diagnostic test menu, apply our multiplexing technology to other infectious diseases, and rapidly expand our product offering using the latest in molecular diagnostic instrumentation platforms and technologies.

We currently have collaborations with research institutes, commercial pathology laboratories and hospitals around the world. Several of our collaborating research institutes have well-annotated clinical specimen banks, providing us additional opportunities to validate new DNA/RNA or other biomarkers to further explore the effective development of personalized medicine.

The involvement of our collaborating partners means a focus on the development of new and commercialization relevant AmpaSand molecular diagnostics and a fast process to market. The expansion into overseas markets is facilitated by our ability to validate our products on a range of molecular diagnostic instrumentation platforms and technologies.

 

New Clinical Studies

We believe well designed clinical studies with well stored and archived samples provide the optimal data for development of our new products. We are currently participating in two significant clinical studies and remain open to further collaborations.