Hello Zahraa A. Alshaheeb,

It is an error. You could either have a false positive or a false negative PCR result.

A false positive is when you determine that something is true when it is actually false, and a false negative is saying something is false when it is actually true.

False negatives can lead to a missed or late diagnosis, putting a patient’s health and survival at risk, while false positives can result in unnecessary additional tests and treatment, including unnecessary costs.

You may avoid false positive results by using strict laboratory hygiene practices, good pipetting techniques, and sterile labware, and the allocation of dedicated PCR stations or rooms. 

The causes of false negatives are wide-ranging and include degraded or insufficient nucleic acid, such as from excessive freeze-thaw cycles; contamination with inhibitors from the environment or DNases and RNases; poorly designed PCR; low-quality reagents; poor or inconsistent techniques during sample processing; and low-quality, faulty, or poorly calibrated equipment. 

For more information on how to prevent false positive and false negative PCR results, you may want to refer to the link below.

https://www.clinicallab.com/preventing-false-positive-and-false-negative-pcr-results-26261

Best.

Any chance the bulb burned out in your QPCR machine? That happened to me! Can you check your sample with standard end-point PCR first?

Amy Klocko, the machine is under control and checked routinely..

It's a good idea to check the end-point.. Thank you.

Malcolm Nobre many thanks for your covered answer for all the points!