Pathogen testing requirements for tomato and capsicum seeds

Some existing seed testing requirements will remain unchanged and continue to apply under the updated protocols. Laboratory test reports for tomato and capsicum seed for export must continue to include the following details:

1. The name and address of the testing laboratory
2. The full botanical name of the species tested
3. The date of testing
4. The type of test done
5. The seed lot number(s)
6. The sample size, which must be 20,000 seeds or 20% of the seed lot for small seed lots
7. The subsample size, which must be no greater than 400 seeds per subsample for all testing conducted by PCR
8. The pathogens targeted
9. The test result confirming freedom from each of the targeted pathogens.

Full testing requirements are available on BICON.

The updated list of approved testing protocols for detecting ToBRFV and ToMMV in tomato and capsicum seed for export to Australia, along with associated requirements, is detailed below:

• Laboratories must use 2 reverse-transcription qPCR protocols to verify the status of ToBRFV and ToMMV in each seed lot.
• For ToBRFV, the CaTa28 protocol must be used as one of the 2 qPCR protocols, and either the CSP1325 protocol or the qs1/p1/qas2 protocol must be used as the second protocol (see Table 1 and Table 2).
• For ToMMV, the CaTa9 protocol must be used as one of the 2 qPCR protocols, and either the CSP1572 protocol or the ToMMV2 protocol must be used as the second protocol (see Table 1 and Table 2).
• References to the approved protocols are listed in Table 3. Please note that the department’s requirements for these protocols and for reporting results may differ from some of the parameters stated in the references.
• No other tests are approved for verifying the status of a seed lot regarding these 2 viruses. Laboratories shall not discount a positive result obtained from an approved qPCR test on the basis of any other test results or any other consideration, except retesting following the ‘Retesting’ section.

• The testing protocols that will no longer be approved for use from 12 November 2025 for detecting ToBRFV and ToMMV are listed in Table 4.

• Extraction and amplification positive controls must be run and should include an RNA target. At least one weak positive control should be used that produces a Ct value of 30 or higher.
• At least one negative control should be run. If any negative controls yield Ct values below 35, the testing should be considered invalid, and the department should be contacted.

• A Ct cut-off value of 35 applies to all qPCR tests for ToBRFV and ToMMV. A statement must be included on laboratory reports that states the Ct cut-off point used to determine the result.
• If 2 or more qPCR replicates are run for a subsample, the lowest Ct value must be used to interpret the result for that subsample.
• If the Ct value of the subsample is equal to or below 35, this will be considered a positive result, i.e. the target virus has been detected.
• If the Ct value of the subsample is above 35, this will be considered a negative result, i.e. the target virus has not been detected.
• If a laboratory considers that further information is relevant to the interpretation of a set of test results for a particular seed lot, the laboratory can contact the department for advice. We will consider such information before making a final decision on the status of the consignment.

A laboratory may retest a qPCR where the result is considered to be potentially anomalous, for example if the amplification curve deviates substantially from a typical sigmoidal shape.

• Retesting must be conducted using the seed homogenate (seed slurry) or RNA extract from the same seed subsample.
• An anomalous or ambiguous qPCR result cannot be superseded by testing a different seed sample or subsample.
• Retesting must include at least 2 additional replicate qPCRs using the same qPCR protocol.
• The results from the retest must be interpreted according to the ‘Ct cut-offs and interpretation requirements’ specified above.
• Any anomalous finding that has not been shown to be negative by retesting based on the interpretation requirements, must be reported as a positive.

• We may conduct retesting of seed lots or trace back investigations of seed lots. Laboratories must provide records of previous testing to assist with these investigations.
• Laboratories must keep copies of the quality control records, protocols used and any validation and verification reports relevant to the tests, and must provide these documents to us if asked.
• Laboratories must keep records of all testing done to meet the Australian requirements for at least 3 years, including Ct values, records of decisions about test results, negative test results, retest results, anomalous results, and results from controls. Images of amplification curves of anomalous results should also be kept.
• If a laboratory resolves an anomalous result and concluded that a seed lot is negative for the virus, the laboratory must record the reason why the anomalous result was considered not to be a detection of the virus.