Stark SARS-CoV-2 Molecular Diagnostic Kit

The Stark Sars-CoV-2 Molecular Diagnostic kit employs a reverse transcription-PCR assay tailored to specifically detect the virus, with its detection facilitated by Real-Time PCR. This kit is designed for the qualitative detection of nucleic acid from SARS-CoV-2 in samples obtained from nasopharyngeal swabs, oropharyngeal (throat) swabs, anterior nasal swabs, nasal washes, and nasal aspirations from individuals suspected of COVID-19 by their healthcare provider. Testing is restricted to laboratories certified under the Clinical Laboratory Improvement Amendment. In adherence to regulations set forth by the Ministry of Health, the sale of COVID-19 diagnostic kits is permitted solely to licensed laboratories. This kit is intended for emergency use and for In Vitro Diagnostic (IVD) purposes. Additionally, the test serves to identify the genome of SARS-CoV-2, which is discernible in samples from patients with acute viral infections. A positive result indicates the presence of the SARS-CoV-2 genome; however, accurate diagnosis necessitates consideration of the patient’s clinical history and other medical conditions. It is imperative to note that positive results do not definitively rule out bacterial infection or co-infection with other viruses. The detected agent may not be the sole causative agent of the disease. Laboratories are obligated to report all positive cases to the relevant public health authorities. Conversely, negative results do not conclusively exclude SARS-CoV-2 infection and should not be the sole basis for patient management decisions. Negative results should be interpreted in conjunction with clinical observations, patient history, and epidemiological data.

The Stark Sars-CoV-2 Molecular Diagnostic kit is a Real-Time reverse transcription-polymerase chain reaction (rRT-PCR) test specifically designed for the detection of RNA from SARS-CoV-2 in respiratory specimens obtained from patients suspected of COVID-19 by their healthcare provider. This kit enables qualitative detection of the RdRp and N genes of SARS-CoV-2 RNA.

With a simple centrifugation and lysis step, the sample mixture can be directly added to the 2019-nCoV-PCR master mix (Q-ROMAX+ RTase+ ProI) for rRT-PCR amplification. An internal control targeting the RNase P gene is incorporated to monitor the entire process, from sample collection to rRT-PCR, ensuring the accuracy of results and minimizing the risk of false-negative outcomes. The Limit of Detection (LoD) of the kit is 100 copies/ml, providing reliable sensitivity for the detection of SARS-CoV-2 RNA in clinical specimens.

Materials Required (but Not Provided)

• Nylon or Dacron swab with an aluminum or plastic shaft.
• DNase-RNase-free microtubes for sampling (1.5ml).
• PCR microtube 0.1ml or 0.2ml strip.
• Various models of pipettes and pipette tips (10µl, 100µl, and 1000µl of filter pipette tips).
• Surface sanitizing solution.
• Disposable Powder-Free gloves and surgical gown.
• Different types of Real-Time PCR Instruments (with green, yellow, and orange channels).
• Centrifuge (capable of reaching 13000 rpm).
• Microcentrifuge.
• Vortex.
• Cool box

Real-Time PCR Instruments

This kit is compatible with the following instruments:

• Rotor-Gene Q, 5plex
• Corbett Rotor-Gene 3000 & 6000
• Mic qPCR Cycler
• ABI StepOne & StepOnePlus
• Biorad CFX96 Real-Time PCR
• Roche LightCycler® 96 Real-Time PCR System
• Anatolia Montania 484 Real-Time PCR Instrument

Sample Collection

The Stark Sars-CoV-2 Molecular Diagnostic kit is designed for the qualitative detection of nucleic acid from SARS-CoV-2 in respiratory specimens. It is crucial to ensure proper collection procedures to minimize the risk of contamination during collection, storage, and transportation. Specimens should be handled with caution and considered potentially infectious, adhering to relevant regulations and biosafety guidelines. Synthetic-tipped swabs, such as nylon or Dacron, with aluminum or plastic shafts are recommended for collection. Cotton swabs with wooden shafts should be avoided. After sampling, swabs should be promptly placed in a suitable virus transport medium.

Storage and Delivery of Specimens

Specimens should ideally be tested within 24 hours if stored at 4°C. For samples that cannot be tested within this timeframe, storage at -70°C or below is recommended. Alternatively, specimens can be stored at -20°C for up to ten days. Nucleic acid extracted from specimens can be stored at -20±5°C for up to 15 days. It is essential to avoid multiple freeze-thaw cycles to maintain sample integrity.

Specimen Isolation

For viral nucleic acid isolation, use a kit which is approved by the Ministry of Health should be used.

Pathogenicity

Coronaviruses can cause a spectrum of illnesses ranging from mild cold-like symptoms to severe respiratory diseases. Symptoms commonly associated with Coronavirus infections include fever, cough, shortness of breath, and respiratory difficulties. Some patients may experience persistent coughing without an apparent cause. In contrast to SARS-CoV, MERS-CoV primarily affects the respiratory system and can lead to complications such as kidney and liver damage. Severe cases of Coronavirus infections may present with additional symptoms such as diarrhea, acute respiratory distress syndrome (ARDS), coagulopathy, and renal failure, necessitating interventions such as hemodialysis.

COVID-19, caused by the novel SARS-CoV-2 virus, typically manifests symptoms within a few days of exposure. However, symptom onset may vary among individuals. Fever is a common symptom, observed in 43.8% of cases upon hospital admission and in 88.7% of hospitalized cases. Dry cough is prevalent in 67.8% of cases, accompanied by respiratory distress, fatigue, and myalgia in 11 to 14% of patients. Diarrhea is reported in 3.8% of cases. The average incubation period for COVID-19 is approximately four to five days. Ground-glass opacity, a characteristic finding on chest CT scans, is observed in 56.4% of cases during the incubation period. Despite this, 17.9% of patients with mild symptoms and 2.9% with severe symptoms may not exhibit abnormalities on radiological imaging. Lymphopenia, characterized by a decreased number of lymphocytes in the blood, is reported in 83.2% of patients upon hospital admission. While some individuals may experience mild or asymptomatic infections, COVID-19 can lead to severe complications such as pneumonia or acute respiratory distress syndrome, particularly in patients with underlying medical conditions, posing a significant risk of mortality.

Transmission of Coronavirus

The SARS-CoV-2 virus can spread through airborne particles produced by coughing or sneezing, similar to the flu. Close contact with infected individuals, particularly in indoor settings, poses a higher risk of transmission. While outdoor environments generally carry a lower risk, prolonged indoor exposure to infected individuals, such as hospitalized COVID patients, can facilitate human-to-human transmission. The exact mode of transmission, whether from animal-to-human or through contaminated surfaces, is still under investigation.

Workstation Preparation

Before commencing work, ensure all work surfaces, pipettes, centrifuges, and other equipment are thoroughly cleaned and sanitized to minimize the risk of nucleic acid contamination. Sanitizers such as 70% Ethanol or 10% Sodium Hypochlorite should be used for effective disinfection.

Protocol

Process

• Remove each component from the diagnostic kit and place them on the bench at room temperature.
• Allow the reagents to equilibrate to room temperature.
• After equilibration, briefly vortex each component to ensure homogeneity for later use.
• Ensure that the isolated sample volume for this test is 10µl.
• Refer to Table 1 to prepare the reaction components (Master Mix) according to the specified quantities.
• Perform Real-Time PCR according to the instructions provided in Table 2.

Table1: Regent’s preparation per one single reaction.

Table 2: PCR program for one-step Multiple Real Time-RT-PCR.

The Stark Sars-CoV-2 Molecular Diagnostic kit undergoes rigorous testing in accordance with standards set by the Clinical and Laboratory Standards Institute and the World Health Organization (WHO). These tests are conducted on a lot-to-lot basis to uphold consistent product quality. For detailed information regarding the results of these tests, please visit http://www.amorph.tech and enter the labeled REF and LOT number in the “Certificate of Analysis” section.

• Perform data analysis for each gene separately using a manual threshold.
• Ensure that the threshold for each sample is within the exponential phase of the fluorescence curves and above any background signal.
• Utilize FAM Fluorophore (green) for the RdRp gene, Texas Red Fluorophore (orange) for the N gene, and HEX Fluorophore (yellow) for the RNase P gene (internal control).
• Employ a negative control as contamination control, ensuring that the fluorescence curve magnitude does not cross the threshold. Ct values less than 35 (Ct<35) indicate possible contamination. Strong signals above 35 in the NTC may indicate PCR artifacts, and in such cases, the shape of the curve should be considered (the S-shaped curve is typical for a positive result).
• Ensure that the internal control or RNase P gene is positive for all clinical specimens with a Ct value of 35 or less, indicating sufficient nucleic acid from the human RNase gene and acceptable sample quality.
• If the internal control curve or RNase P gene Ct value is greater than 40 or absent, it indicates low sample concentration or inhibitors in the reaction. In such cases, the isolated sample is recommended to be diluted by at least half. If the test result is not acceptable upon retest, obtain a new sample from the patient, and repeat the test.
• A positive clinical specimen should have a Ct value of ≤40 for genes or should exhibit positivity for two genes.
• If the expected positive reaction is not achieved (typical S-shaped curve), the performed test is deemed unacceptable, and the test must be repeated while accurately following the kit instructions.
• Identify the reason for the failure of the positive control, take corrective action, and document the results of the corrective action.

For information about these bellow subjects refer to the product IFU:

• Limit of Detection (LoD) – Analytical Sensitivity
• Clinical Sensitivity
• Cross-reactivity (Analytical Specificity)
• Cross-reactivity (Clinical Specificity)
• Accuracy
  • Intra-assay
  • Inter-assay
• Clinical Evaluation

• A false-negative result may occur due to low titration of the virus in the patient sample, improper transportation, and poor quality of sample isolation.
• The genetic diversity of the coronavirus genome can lead to poor primer/probe binding to the target sequence, resulting in false-negative results, despite attempts to design primers or probes for conserved viral genome regions.
• All controls must be verified before result interpretation. Invalid controls render the patient’s results uninterpretable. The diagnostic limit of this kit is Ct≤40, and users must review the fluorescence curve before final interpretation, ensuring all positive curves exhibit an amplification peak.
• Failure to adhere to proper storage conditions for the kit can lead to false-negative results.
• Proper handling of this kit requires experienced and trained personnel. Errors by personnel may lead to invalid results.
• Results obtained from this diagnostic kit are only acceptable when combined with clinical evidence for diagnosing SARS-CoV-2. Definitive diagnosis and treatment of patients should be based on a combination of this test with other test results, medical records, and response to treatment.

Storage

All components of the Stark MTB Molecular Diagnostic Kit are pre-prepared and ready for immediate use upon arrival. Upon receipt, it is recommended to store all reagents at temperatures ranging from -15°C to -30°C. These conditions ensure stability and maintain the integrity of the components until the expiration date indicated on the label.

Warning and Precautions

• Please adhere to laboratory safety protocols.
• Prior to use, thoroughly review the guidelines provided.
• Note that all patient samples and positive controls carry the potential for infectiousness.
• Refrain from eating, drinking, smoking, chewing gum, applying cosmetics, or taking medication while handling hazardous materials and human samples in laboratories. Treat all patient samples and positive controls as potentially infectious.
• Before commencing work, familiarize yourself with all safety guidelines related to handling COVID-19 samples, accessible at https://www.cdc.gov/coronavirus/2019-nCoV/lab-biosafety-guidelines.html.
• The Stark Sars-CoV-2 Molecular Diagnostic kit is intended for use by qualified and trained clinical laboratory personnel who have received specific instruction and training in real-time PCR and in vitro diagnostic procedures. All steps of the procedure, including sampling, storage, shipping, and laboratory tests, must adhere to biosafety and laboratory information management system (LIMS) protocols.
• This test requires a separate and dedicated workstation within the laboratory:
  • Location 1: Preparation area – for assembling test components.
  • Location 2: Sample processing area – for sample isolation and handling.
  • Location 3: Amplification area – for conducting Real-Time PCR tests.
• Clinical laboratories must be equipped with instruments and personnel in accordance with guidelines from the Ministry of Health.
• The contents provided in the kit are specific to COVID-19 testing. Altering or substituting any kit contents may compromise product performance.
• Before commencing tests, ensure that each component is thawed, vortexed, and briefly centrifuged. Avoid subjecting components to repeated freeze-thaw cycles.
• All pipette tips and microtubes must be sterile and free from DNase and RNase contamination. Use filter pipette tips to prevent contamination, and change tips after adding any substances or samples.
• Dispose of hazardous and biological waste in compliance with local and national regulations to prevent environmental contamination. Use decontaminants such as 10% sodium hypochlorite, 70% ethanol, and Surface sanitizing solution for nucleic acid contaminations. Avoid exposing PCR-COVID 19 combinations to sunlight.
• Positive results must be promptly reported to health authorities.