UTI panel real time PCR detection with ABR (100 test)

UTI panel real time PCR detection with ABR (100 test)

Cat. No.: BMG-QP2158-01
Size: 100 tests


Description

Culture-based methods for the detection of UTI pathogens lack sensitivity, are subjective, and prolonged Turnaround Times (48-72 Hours). Clinicians frequently treat patients empirically resulting in poor antibiotic stewardship and increased rate of antibiotic resistance. Our new real-time PCR solution for UTI investigations enables rapid detection without the need of culture— while enabling increased sensitivity and specificity of detection of urinary tract pathogens and keeping cost per sample low. Our real time PCR assays use DNA isolated from specimens such as urine or blood. The product is intended for use by qualified laboratory personnel well trained in nucleic acid amplification techniques and in vitro diagnostic procedures. Positive results do not rule out bacterial infection or co-infection with other pathogens. Negative results do not preclude microbial infection and should not be used as the sole basis for patient management decisions. Test results must be combined with clinical observations, patient history, and epidemiological information.


Summary and explanation of the test

The UTI Pathogen Detection Assay is a real-time PCR test intended for the qualitative detection of DNA from the bacteria and fungus in urine and blood samples.
The assay includes the following urinary tract bacteria targets:
Enterobacter cloacae
Enterococcus faecalis
Escherichia coli
Klebsiella oxytoca
Klebsiella pneumoniae
Morganella morganii
Mycoplasma hominis
Proteus mirabilis
Providencia stuartii
Pseudomonas aeruginosa
Serratia marcescens
Staphylococcus aureus
Staphylococcus saprophyticus
Streptococcus agalactiae
Ureaplasma parvum serovar and Ureaplasma urealyticum
C. albicans; C. glabrata; C. parapsilosis and C. tropicalis
and drug resistance gene:
MecA, MecC, VenA, VenC, KPC and CTX-M


Principle

The primers and probes are selected from the regions of targeted conserved regions of the above bacteria. The primer/probe sets are designed for specific detection of above regions are separated into 4 multiplex qPCR setups. An additional primer/probe set to detect human TERT gene (as an internal control) is also included. DNA extracted from respiratory specimens is amplified in a Real-Time PCR Thermocycler (such as Applied Biosystems 7500 Real-Time PCR Instrument). The fluorescent signal is emitted when the 5’ nuclease active domain of Taq polymerase degrades the probe anneals to target sequence between the forward and the reverse primers. During each cycle, the fluorescence intensity is increased with the additional reporter dye molecules released from their respective probes. The UTI) Pathogen Detection Assay also contains a positive control (Plasmid DNA) and a negative control for testing process monitoring.


Reagents and materials provided
 

Item	Component	Quantity		Volume /T
		100 Tests / Box	500 Tests / Box
1	UTI PCR Mix	1.5 mL/ tube each	7.5 mL/tube each	14 µL
2	Enzyme Mix	550 µL	5x550 µL	1 µL
3	UTI Positive Control	50 μL/ tube × 1	250 μL/ tube × 1	5 μL
4	UTI Negative Control	50 μL/ tube × 1	250 μL/ tube × 1	5 μL

Materials may required but not provided

1. Real-Time PCR Thermocycler compatible with JOE (VIC or HEX is equivalent to JOE), FAM and Cy5 channels.
2. Specimen Collection Containers
3. DNA extraction reagent
4. PCR tubes compatible with Thermocycler.
5. 1.5 mL centrifuge tubes (RNase-Free)
6. Vortex
7. High-speed centrifuge
8. Micropipette (0.5-2 µL, 1-10 µL)
9. Pipette tips with filters
10. Personal protective material

Storage instructions

Store the kit frozen at -20±5°C and avoid repeated freeze-thaw cycles (less than 5 frozen-thaw times allowed). The product is valid for 12 months, and the expiry date of the product is shown on the label.

Specimen type and preservation

Specimen type: urine or blood samples
Specimen preservation: process specimens for bacterial and fugus NA extraction and nucleic acid detection as soon as possible. Specimens can be stored at 4°C for 24 hours, or frozen at -80°C for longer storage, if not processed immediately. Avoid repeated freeze-thaw cycles during transport and storage of the specimens.

Assay PCR reaction setup (at reagent preparation area)

A. Reagent Preparation
To calculate the number (n) of PCR reaction tubes (n= number of specimens to be tested + number of positive controls + number of negative controls), add n x14 μL of SARS-CoV-2 PCR Mix and n x 1 μL of RT-PCR Enzyme Mix to a clean tube, vortex to mix well, centrifuge and aliquot 15 µL of the mix into each PCR reaction tube.

B. Addition of Bacterial DNA
Add 5 μL of bacteria DNA extracts of the specimen or positive/negative control to each PCR tube according to the reaction preparation table below for testing. Then immediately cap the tube tightly, briefly spin the mixture and move it to the amplification area for PCR detection.

• PCR Reaction Preparation

C. PCR amplification (at Amplification Area, use Applied Biosystems 7500 Real-Time PCR System as an example)

1. 1. Place the complete PCR reaction tubes into the fluorescent quantitative PCR analyzer and label positive control, negative control and testing specimen on the Thermocycler software.
2. 2. Enter the PCR reaction volume: 20 μL.
3. 3. Select fluorescence detection channels: JOE, FAM and Cy5 channels. Cy5 is the E gene indicator channel, FAM is the ORF1ab indicator channel, and JOE is the RNase P gene indicator channel (internal control).
4. 4. Select PCR cycle parameter setting following the instructions below, save the file and start the PCR run.
5. 5. Analyze the results according to your thermocycler manufacturer’s instructions. If the positive and negative control samples do not meet their respective requirements, the PCR run must be repeated.

Determination of results

1. The results can only be determined when the results of both controls meet their requirements.
2. Criterion for a positive specimen: a specimen can be reported as positive when FAM, Cy5 and JOE channels are all positive.
3. Criterion for a negative specimen: a specimen can be reported as negative when the JOE channel is positive and the FAM and Cy5 channels are negative

Compatibility of real-time PCR thermocyclers
Product performance

Product analysis performance evaluation results:

1. The analytical sensitivity of this kit is 200 copies/mL.