TriCon 2023 Exhibit Poster "From Urine Samples to Diagnosis"

FROM URINE SAMPLES TO DIAGNOSIS: FASTER RESULTS FOR PATHOGEN MOLECULAR DETECTION

Podaru M.N, Ph. D. ; Leonardia S., Radic-Hunter S., Bansair E., Fouqueau T, Ph. D. ; Amasio M. , Ph. D.

www.meridianbioscience.com/lifescience

Introduction

Urine is an ideal diagnostic specimen due to its non-invasive nature and ease of collection and is widely used in the clinical diagnosis of urinary tract infections (UTIs) and sexually transmitted diseases (STDs). In this work, we demonstrate how our new molecular chemistries can support alternative methods to the traditional urine cell culture approach by shortening the time to results and reducing costs. Our qPCR and LAMP Urine-Specific master mixes are compatible with lyophilization or air-drying and do not require purified nucleic acids. This bypasses time-consuming and costly nucleic acid purification step, making them ideal for POC applications. The qPCR/RT-qPCR mixes tolerate high concentrations of the sample (>40% human urine) while maintaining sensitivity down to 1 copy/reaction and high multiplexing capacity. The LAMP/RT-LAMP mixes can provide results in as little as 20 minutes while detecting pathogens down to 1 copy directly from urine. Furthermore, as our data shows, these new chemistries have great potential in the future diagnosis of vector-borne tropical diseases directly from urine samples. These innovative urine-specific chemistries have the unique ability to increase testing throughput and shorten turnaround times while maintaining high sensitivity.

Urine-Optimized Master Mix

Each component was optimized for the development of Urine-specific master mixes.

Materials and Methods

Reporting (up to 3 days)

Culture test

Antibiotic susceptibility test

Urine sample: Gender Unspecified, Unfiltered, 5-Donor Pooled Human Urine (BioIVT) was used in this study qPCR/RT-qPCR reactions:

Reporting (up to 5 hours)

Nucleic-acid Extraction

Standard qPCR

Urine-specific qPCR/RT-qPCR Mixes, Lyo-Ready ™ Direct DNA qPCR Urine (MDX152) and Lyo-Ready ™ Direct RNA/DNA qPCR Urine (MDX153) from Meridian Bioscience, were lyophilised with primers and probes according to the product’s technical specifications and stored in a sealed pouch with silica. The qPCR/RT-qPCR reactions were initiated by the addition of target-containing pooled urine sample and run using the cycling conditions as described below: • qPCR cycle: 95°C for 3 mins; 45 cycle of 95°C for 10 sec and 65°C for 25 sec • RT-qPCR cycle: 50°C for 10 mins; 95°C for 2 mins; 45 cycle of 95°C for 10 sec and 65°C for 30 sec ToughMix PCR Master Mix (Quantabio), KAPA Probe Force qPCR kit (Roche), Reliance One-Step Multiplex RT-qPCR Supermix ™ (Bio-Rad) and TaqPath ™ ProAmp ™ Multiplex Master Mix (ThermoFisher) in liquid format were tested in the same reactions using the concentrations recommended by the suppliers. LAMP/RT-LAMP reactions: Urine-specific LAMP/RT-LAMP Mixes, Lyo-Ready ™ Direct DNA LAMP Urine (MDX154) and Lyo-Ready ™ Direct RNA/DNA LAMP Urine (MDX155) from Meridian Bioscience, were lyophilised with primers and probes according to the product’s technical specifications and stored in a sealed pouch with silica. The LAMP/RT-LAMP reactions were initiated by the addition of target-containing pooled urine sample and run using the cycling conditions as described below: • LAMP/RT-LAMP cycle: 65°C for 60 mins. WamStart ® LAMP kit (DNA&RNA) (NEB) and SuperScript ™ IV RT-LAMP Master Mix (ThermoFisher) in liquid format were tested in the same reactions using the master mix concentration recommended by the suppliers.

Reporting (< 2 hours)

Direct qPCR

Comparison of workflows of urine specimens to different pathogen detection tests. The traditional culture-based methods are performed using manual methods and take up to 3 days to identify the microbial pathogens responsible for the infection. Standard real-time PCR approaches could deliver the results in 5 hours, but required long, laborious and expensive DNA/RNA extraction steps. Urine-specific chemistries enable the detection of pathogens directly from urine, significantly reducing the turnaround time and cost of molecular tests.

Chemical biomarkers

Urine Culture

Standard qPCR Urine-specific chemistries

High High

Low Low

Variable Variable Possible

Good Good Good

Specificity Sensitivity

Yes No Yes

No Yes No

Polymicrobial infection detection Additional confirmation requirements

No No

Compatibility with POCT Detection turnaround time

< 2 hours

Instant

1-3 days

< 5 hours

Table 1. Characteristics of available urine testing methods. Regardless of the microbial/viral targets, urine-specific master mixes will allow to you have results in 2 hours from the sample collection at the testing lab. The urine-specific master mixes allow you to detect multiple pathogen down to 1-copy directly from urine.

qPCR

High Multiplexing Capacity for Urinary Tract Infection Testing

LAMP

Rapid and Sensitive Detection of Sexually Transmitted Diseases

Figure 1. Direct multiplexed detection of four Urinary Tract Infection targets from urine. Four pathogens ( Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Staphylococcus saprophyticus ) were detected in a quadruplex reaction that contained 40% pooled human urine using lyophilized MDX152, Quantabio, Roche and ThermoFisher mixes. MDX152 showed the earliest Ct and highest end-fluorescence in detecting all four targets directly from urine compared to the other mixes. The data demonstrate that the urine-specific qPCR mix is ideal for robust multiplex UTI tests.

Sample

NTC

Sample

NTC

Sample

NTC

Sample

NTC

Sample

NTC

1000

100

NTC

N. gonorrhoeae L. interrogans M. genitalium T. vaginalis

HSV1

qPCR

High Sensitivity for Sexually Transmitted Disease Detection

Figure 4. Rapid and Sensitive detection of sexually transmitted infections. A) Lyophilized MDX154 was tested in LAMP reactions against NEB and ThermoFisher mixes for the detection of pathogens including Neisseria gonorrhoeae, Leptospira interrogans, Mycoplasma genitalium, Trichomonas vaginalis and Herpes Simplex Virus 1 (HSV1). Error bars represent the standard deviation across four technical replicates. B) The ability of the above LAMP mixes to amplify a 10-fold dilution series of T. vaginalis genomic DNA (1000, 100, 10 and 1 copy/reaction) was tested in the presence of 20% pooled human urine. Error bars represent the standard deviation across four technical replicates. MDX154 provides the fastest and the most sensitive detection of DNA STD targets (Sample) among the tested LAMP mixes. It also has the highest specificity by having the earliest TTR but delayed no-template control (NTC) amplification signals across a broad range of STD targets. The data demonstrate that the urine-specific LAMP mix is ideal for rapid UTI and STD tests.

RT-LAMP

Rapid and Specific Detection of Mosquito-borne Infectious Diseases

Figure 2. Direct multiplexed detection of four Sexually Transmitted Infection targets from urine A) Four pathogens ( Mycoplasma genitalium, Treponema pallidum (Syphilis), Chlamydia trachomatis and Neisseria gonorrhoeae ) were detected in a quadruplex reaction that contained 20% pooled human urine using lyophilized MDX152, Quantabio, Roche and ThermoFisher mixes. B) The ability of the above qPCR mixes to amplify a 10-fold dilution series of syphilis DNA (1000, 100, 10 and 1 copy/reaction) was tested in the presence of 10% pooled human urine. MDX152 showed the earliest Ct and highest end-fluorescence than the other mixes on all four targets. The data demonstrate that the urine-specific qPCR mix has the best performance and multiplexing capabilities, and was the only mix capable of detecting 1-copy of pathogen DNA in reaction directly from urine, making it ideal for sensitive multiplex STD test.

Figure 5. Fast Detection of Mosquito-borne Infectious Diseases. Lyophilized MDX155 was lyophilized with primers and then tested in RT-LAMP reactions against NEB and ThermoFisher mixes for the detection of RNA viruses including Chikungunya (CHIKV), Hepatitis C (HCV), West Nile (WNV), Zika (ZIKV) or Yellow Fever (YFV). Error bars represent the standard deviation across three technical replicates. MDX155 provides the fastest and the most specific detection of viral targets responsible for mosquito-born infectious diseases, making it ideal for rapid tropical disease tests.

RT-qPCR

Highly Sensitive Detection of Mosquito-borne Infectious Diseases

Sample

NTC

Sample

NTC

Sample

NTC

Sample

NTC

Sample

NTC

Sample

NTC

CHIKV

DENV

HCV

WNV

ZIKV

YFV

Conclusions

• U rine-Specific Master Mixes can detect DNA and RNA targets down to 1-copy directly from both crude urine samples and their purified extracts • U rine-specific qPCR Mixes can detect multiple pathogens from both crude urine samples and their extracts in a single reaction, allowing polymicrobial infection detection • U rine-specific LAMP Mixes allow rapid (<20 min) and sensitive (down to 1-copy/reaction) detection of targets directly from both crude urine samples and their extracts • U rine-specific qPCR and LAMP Mixes are compatible with lyophilization and air-drying , making them ideal for Point-of-Care Testing (POCT) • B y bypassing the nucleic-acid extraction steps , urine-specific master mixes can significantly reduce the cost and the turnaround time of urine testing

Figure 3. Direct multiplexed detection of four Mosquito-borne Infection targets from urine A) Three arbovirus (Dengue, Zika and Chikungunya) and one protozoan parasite ( Plasmodium falciparum (Malaria)) were detected in a quadruplex reaction that contained 20% pooled human urine using lyophilized MDX153, Quantabio, Bio-Rad and ThermoFisher mixes. B) The ability of the above master mixes to amplify a 10-fold dilution series of Zika RNA (1000, 100, 10 and 1 copy/reaction) was tested in the presence of 5% pooled human urine. MDX153 showed the earliest Ct and highest end fluorescence than the other mixes on all four targets. The data demonstrate that the urine-specific RT-qPCR mix has the best performance and multiplexing capabilities, and was capable of detecting 1-copy of viral RNA in reaction directly from urine, making it ideal for sensitive multiplex tropical-disease tests.

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